Engineered imine reductases and methods for the reductive amination of ketone and amine compounds

ABSTRACT

The present application provides engineered polypeptides having imine reductase activity, polynucleotides encoding the engineered imine reductases, host cells capable of expressing the engineered imine reductases, and methods of using these engineered polypeptides with a range of ketone and amine substrate compounds to prepare secondary and tertiary amine product compounds.

The present application is a Divisional of U.S. patent application Ser.No. 15/519,949, filed Apr. 18, 2017, which is a national stageapplication filed under 35 USC § 371 and claims priority tointernational application to PCT International Application No.PCT/US2015/062300, filed Nov. 24, 2015, which claims priority to U.S.Prov. Pat. Appln. Ser. No. 62/084,037, filed Nov. 25, 2014, each ofwhich are hereby incorporated by reference, in their entireties and forall purposes.

TECHNICAL FIELD

The invention relates to engineered polypeptides having imine reductaseactivity useful for the conversion of various ketone and aminesubstrates to secondary and tertiary amine products.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX2-145WO2_ST25_Substitute_Seq_2.txt”, a creation date ofJan. 24, 2019, and a size of 3,008 kilobytes. The Sequence Listing filedvia EFS-Web is part of the specification and is incorporated in itsentirety by reference herein.

BACKGROUND

Chiral secondary and tertiary amines are important building blocks inpharmaceutical industry. There are no efficient biocatalytic routesknown to produce this class of chiral amine compounds. The existingchemical methods use chiral boron reagents or multi step synthesis.

There are a few reports in the literature of the biocatalytic synthesisof secondary amines Whole cells of the anaerobic bacteriumAcetobacterium woodii imine reductase activity was reported to reducebenzylidine imines and butylidine imines (Chadha et al., Tetrahedron:Asym., 19:93-96 [2008]). Another report uses benzaldehyde orbutyraldehyde and butyl amine or aniline in aqueous medium using wholecells of Acetobacterium woodii (Stephens et al., Tetrahedron 60:753-758[2004]). Streptomyces sp. GF3587 and GF3546 were reported to reduce2-methyl-1-pyrroline stereoselectively (Mitsukara et al., Org. Biomol.Chem., 8:4533-4535 [2010]).

One challenge in developing a biocatalytic route for this type ofreaction is the identification of an enzyme class that could beengineered to provide to carry out such reactions efficiently underindustrially applicable conditions. Opine dehydrogenases are a class ofoxidoreductase that act on CH—NH bonds using NADH or NADPH as co-factor.A native reaction of the opine dehydrogenases is the reductive aminationof α-keto acids with amino acids. At least five naturally occurringgenes having some homology have been identified that encode enzymeshaving the characteristic activity of opine dehydrogenase class. Thesefive enzymes include: opine dehydrogenase from Arthrobactor sp. strain1C (CENDH); octopine dehydrogenase from Pecten maximus (great scallop)(OpDH); ornithine synthase from Lactococcus lactis K1 (CEOS); β-alanineopine dehydrogenase from Cellana grata (BADH); and tauropinedehydrogenase from Suberites domuncula (TauDH). The crystal structure ofthe opine dehydrogenase CENDH has been determined (See e.g., Britton etal., Nat. Struct. Biol., 5: 593-601 [1998]). Another enzyme, N-methylL-amino acid dehydrogenase from Pseudomonas putida (NMDH) is known tohave activity similar to opine dehydrogenases, reacting with α-ketoacids and alkyl amines, but appears to have little or no sequencehomology to opine dehydrogenases and amino acid dehydrogenases. NMDH hasbeen characterized as belonging to a new superfamily of NAD(P) dependentoxidoreductase (See e.g., U.S. Pat. No. 7,452,704 B2; and Esaki et al.,FEBS J., 272, 1117-1123 [2005]).

There is a need in the art for biocatalysts and processes for usingthem, under industrially applicable conditions, for the synthesis ofchiral secondary and tertiary amines.

SUMMARY

The present invention provides novel biocatalysts and associated methodsto use them for the synthesis of chiral secondary and tertiary amines bydirect reductive amination using an unactivated ketone and anunactivated amine as substrates. The biocatalysts of the invention areengineered polypeptide variants derived by directed evolution of theengineered enzymes of SEQ ID NO: 4,6 and 8, which in turn had beengenerated by directed evolution of an initial wild-type gene fromArthrobacter sp. strain 1C which encodes an opine dehydrogenase havingthe amino acid sequence of SEQ ID NO:2. These engineered polypeptidesare capable of catalyzing the conversion of a ketone (includingunactivated ketone substrates such as cyclohexanone and 2-pentanone) oraldehyde substrate, and a primary or secondary amine substrate(including unactivated amine substrates such as butylamine, aniline,methylamine, and dimethylamine) to form a secondary or tertiary amineproduct compound. The enzymatic activity of these engineeredpolypeptides derived from opine dehydrogenases is referred to as “iminereductase activity,” and the engineered enzymes disclosed herein arealso referred to, as “imine reductases” or “IREDs.” The general iminereductase activity of the IREDs is illustrated below in Scheme 1.

The engineered polypeptides having imine reductase activity of thepresent invention can accept a wide range of substrates. Accordingly, inthe biocatalytic reaction of Scheme 1, the R¹ and R² groups of thesubstrate of formula (I) are independently selected from a hydrogenatom, or optionally substituted alkyl, alkenyl, alkynyl, alkoxy,carboxy, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl,arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; and the R³and R⁴ groups of the substrate of formula (II) are independentlyselected from a hydrogen atom, and optionally substituted alkyl,alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl,alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl,heteroaryl, and heteroarylalkyl, with the proviso that both R³ and R⁴cannot be hydrogen. Optionally, either or both of the R¹ and R² groupsof the substrate of formula (I) and the R³ and R⁴ groups of thesubstrate of formula (II), can be linked to form a 3-membered to10-membered ring. Further, the biocatalytic reaction of Scheme 1 can bean intramolecular reaction wherein at least one of the R¹ and R² groupsof the compound of formula (I) is linked to at least one of the R³ andR⁴ groups of the compound of formula (II). Also, either or both of thecarbon atom and/or the nitrogen indicated by * in the product compoundof formula (III) can be chiral. As described further herein, theengineered polypeptides having imine reductase activity exhibitstereoselectivity, thus, an imine reductase reaction of Scheme 1 can beused to establish one, two, or more, chiral centers of a productcompound of formula (III) in a single biocatalytic reaction.

In some embodiments, the present invention provides an engineeredpolypeptide comprising an amino acid sequence having at least 80%sequence identity to an amino acid reference sequence of SEQ ID NOS:2,4, or 6, and further comprising one or more amino acid residuedifferences as compared to the reference amino sequence, wherein theengineered polypeptide has imine reductase activity. In some embodimentsof the engineered polypeptide, the imine reductase activity is theactivity of Scheme 1, optionally, a reaction as shown in Table 2.

Additionally, as noted above, the crystal structure of the opinedehydrogenase CENDH has been determined (See e.g., Britton et al., Nat.Struct. Biol., 5: 593-601 [1998]). Accordingly, this correlation of thevarious amino acid differences and functional activity disclosed hereinalong with the known three-dimensional structure of the wild-type enzymeCENDH can provide the ordinary artisan with sufficient information torationally engineer further amino acid residue changes to thepolypeptides provided herein (and to homologous opine dehydrogenaseenzymes including OpDH, BADH, CEOS, and TauDH), and retain or improve onthe imine reductase activity or stability properties. In someembodiments, it is contemplated that such improvements can includeengineering the engineered polypeptides of the present invention to haveimine reductase activity with a range of substrates and provide a rangeof products as described in Scheme 1.

In some embodiments, the present invention provides an engineeredpolypeptide comprising an amino acid sequence with at least 80% sequenceidentity to a reference sequence of SEQ ID NO:2 and at least onesubstitution at a position selected from X9, X12, X31, X37, X44, X46,X48, X55, X56, X65, X82, X93, X98, X108, X137, X138, X141, X142, X143,X153, X154, X155, X156, X159, X166, X167, X168, X173, X177, X178, X184,X185, X195, X197, X198, X199, X200, X201, X203, X205, X206, X210, X213,X215, X216, X218, X220, X221, X223, X224, X226, X234, X239, X242, X245,X251, X253, X256, X257, X259, X260, X261, X262, X263, X265, X266, X267,X268, X269, X272, X273, X274, X276, X277, X278, X279, X280, X281, X282,X283, X284, X285, X289, X290, X291, X292, X293, X294, X330, and X349,wherein the polypeptide has imine reductase activity as compared to SEQID NO:2. In some embodiments, the amino acid sequence of the engineeredpolypeptide comprises at least one residue difference as compared to thereference sequence of SEQ ID NO:4, selected from X198, X153, X167, X265,X262, X108, X234, X284, X282, X220, X272, X256, X267, X242, X281, X197,X277, X224, and X143. In some additional embodiments, the amino acidsequence of the engineered polypeptide comprises at least one residuedifference as compared to the reference sequence of SEQ ID NO:6,selected from: X283, X262, X9, X259, X220, X267, X153, X279, X200, X224,X256, X137, X143, X260, X261, X154, X276, and X185. In some furtherembodiments, the amino acid sequence of the engineered polypeptidecomprises at least one residue difference as compared to the referencesequence of SEQ ID NO:8, selected from: X12, X31, X37, X44, X46, X48,X55, X56, X65, X82, X93, X98, X108, X137, X138, X141, X142, X143, X153,X154, X155, X156, X159, X166, X168, X173, X177, X178, X184, X185, X195,X197, X199, X200, X201, X203, X205, X206, X210, X213, X215, X216, X218,X220, X221, X223, X226, X234, X239, X242, X245, X251, X253, X256, X257,X259, X260, X261, X262, X263, X265, X266, X267, X268, X269, X273, X274,X277, X278, X279, X280, X281, X282, X283, X284, X285, X289, X290, X291,X292, X293, X294, X330, and X349.

The present invention further provides engineered imine reductasepolypeptides, wherein the imine reductase activity comprises convertingat least one of the following ketone and amine substrate compound pairsto the listed amine product compound under suitable reaction conditions:(a) ketone substrate compound (1a) and amine substrate compound (1a) toproduce compound (3a); (b) ketone substrate compound (1b) and aminesubstrate compound (2b) to produce compound (3b); (c) ketone substratecompound (1c) and amine substrate compound (2c) to produce compound(3c); (d) ketone substrate compound (1d) and amine substrate compound(2d) to produce compound (3d); (e) ketone substrate compound (1d) andamine substrate compound (2c) to produce compound (3e); (f) ketonesubstrate compound (1d) and amine substrate compound (2c) to producecompound (3f); and ketone substrate compound (1d) and amine substratecompound (2c) to produce compound (3g). In some embodiments, the iminereductase activity involved in converting the ketone and amine substratecompound pair to the listed amine product compound under suitablereaction conditions is increased at least 2-fold as compared to thecorresponding activity of the reference polypeptide of SEQ ID NOS:4, 6,12, 30, 126, 173, 360, and/or 550.

The present invention also provides engineered polypeptides in which theamino acid sequence comprises a sequence selected from the groupconsisting of the even-numbered sequence identifiers of SEQ IDNOS:4-1300.

The present invention also provides polynucleotides encoding theengineered polypeptides provided herein. In some embodiments, thepolynucleotide comprises a nucleic acid sequence selected from the groupconsisting of the odd-numbered sequence identifiers of SEQ IDNOS:5-1299.

The present invention also provides expression vectors comprising thepolynucleotides provided herein. The present invention also provideshost cells comprising at least one polynucleotide provided herein. Thepresent invention further provides host cells comprising at least oneexpression vector as provided herein.

The present invention also provides methods of preparing the engineeredpolypeptide having imine reductase activity, comprising culturing a hostcell provided herein, under conditions suitable for expression of thepolypeptide, optionally further comprising isolating the engineeredpolypeptide.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly indicates otherwise. Thus, for example, reference to “apolypeptide” includes more than one polypeptide. Similarly, “comprise,”“comprises,” “comprising” “include,” “includes,” and “including” areinterchangeable and not intended to be limiting. It is to be understoodthat where descriptions of various embodiments use the term“comprising,” those skilled in the art would understand that in somespecific instances, an embodiment can be alternatively described usinglanguage “consisting essentially of” or “consisting of.” It is to befurther understood that where descriptions of various embodiments usethe term “optional” or “optionally” the subsequently described event orcircumstance may or may not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not. It is to be understood that both the foregoing generaldescription, and the following detailed description are exemplary andexplanatory only and are not restrictive of this invention. The sectionheadings used herein are for organizational purposes only and not to beconstrued as limiting the subject matter described.

Abbreviations

The abbreviations used for the genetically encoded amino acids areconventional and are as follows:

Amino Acid Three-Letter One-Letter Abbreviation Alanine Ala A ArginineArg R Asparagine Asn N Aspartate Asp D Cysteine Cys C Glutamate Glu EGlutamine Gln Q Glycine Gly G Histidine HIS H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the amino (N) to carboxy (C)direction in accordance with common convention.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleotides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When nucleic acid sequences are presented as astring of one-letter abbreviations, the sequences are presented in the5′ to 3′ direction in accordance with common convention, and thephosphates are not indicated.

Definitions

In reference to the present invention, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings:

“Protein”, “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Polynucleotide” or “nucleic acid’ refers to two or more nucleosidesthat are covalently linked together. The polynucleotide may be whollycomprised ribonucleosides (i.e., an RNA), wholly comprised of 2′deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and 2′deoxyribonucleosides. While the nucleosides will typically be linkedtogether via standard phosphodiester linkages, the polynucleotides mayinclude one or more non-standard linkages. The polynucleotide may besingle-stranded or double-stranded, or may include both single-strandedregions and double-stranded regions. Moreover, while a polynucleotidewill typically be composed of the naturally occurring encodingnucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), itmay include one or more modified and/or synthetic nucleobases, such as,for example, inosine, xanthine, hypoxanthine, etc. Preferably, suchmodified or synthetic nucleobases will be encoding nucleobases.

“Opine dehydrogenase activity,” as used herein, refers to an enzymaticactivity in which a carbonyl group of a 2-ketoacid (e.g., pyruvate) andan amino group of a neutral L-amino acid (e.g., L-norvaline) areconverted to a secondary amine dicarboxylate compound (e.g., such asN-[1-(R)-(carboxy)ethyl]-(S)-norvaline).

“Opine dehydrogenase,” as used herein refers to an enzyme having opinedehydrogenase activity. Opine dehydrogenase includes but is not limitedto the following naturally occurring enzymes: opine dehydrogenase fromArthrobacter sp. strain 1C (CENDH) (SEQ ID NO:2);

“Imine reductase activity,” as used herein, refers to an enzymaticactivity in which a carbonyl group of a ketone or aldehyde and an aminogroup a primary or secondary amine (wherein the carbonyl and aminogroups can be on separate compounds or the same compound) are convertedto a secondary or tertiary amine product compound, in the presence ofco-factor NAD(P)H, as illustrated in Scheme 1.

“Imine reductase” or “IRED,” as used herein, refers to an enzyme havingimine reductase activity. It is to be understood that imine reductasesare not limited to engineered polypeptides derived from the wild-typeopine dehydrogenase from Arthrobacter sp. strain 1C, but may includeother enzymes having imine reductase activity, including engineeredpolypeptides derived from other opine dehydrogenase enzymes, such asoctopine dehydrogenase from Pecten maximus (OpDH), ornithine synthasefrom Lactococcus lactis K1 (CEOS), β-alanopine dehydrogenase fromCellana grata (BADH), tauropine dehydrogenase from Suberites domuncula(TauDH); and N-methyl L-amino acid dehydrogenase from Pseudomonas putida(NMDH); or an engineered enzyme derived from a wild-type enzyme havingimine reductase activity Imine reductases as used herein includenaturally occurring (wild-type) imine reductase as well as non-naturallyoccurring engineered polypeptides generated by human manipulation.

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” or “engineered” or “non-naturally occurring” when usedwith reference to, e.g., a cell, nucleic acid, or polypeptide, refers toa material, or a material corresponding to the natural or native form ofthe material, that has been modified in a manner that would nototherwise exist in nature, or is identical thereto but produced orderived from synthetic materials and/or by manipulation usingrecombinant techniques. Non-limiting examples include, among others,recombinant cells expressing genes that are not found within the native(non-recombinant) form of the cell or express native genes that areotherwise expressed at a different level.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence for optimal alignment of the two sequences. Thepercentage may be calculated by determining the number of positions atwhich the identical nucleic acid base or amino acid residue occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Alternatively, the percentage may becalculated by determining the number of positions at which either theidentical nucleic acid base or amino acid residue occurs in bothsequences or a nucleic acid base or amino acid residue is aligned with agap to yield the number of matched positions, dividing the number ofmatched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Those of skill in the art appreciate that there aremany established algorithms available to align two sequences. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482(1981), by the homology alignment algorithm of Needleman and Wunsch, J.Mol. Biol., 48:443 (1970), by the search for similarity method ofPearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package), or by visualinspection (see generally, Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1995Supplement) (Ausubel)). Examples of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.,J. Mol. Biol., 215: 403-410 (1990) and Altschul et al., Nucl. AcidsRes., 3389-3402 (1977), respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix(See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915[1989]). Exemplary determination of sequence alignment and % sequenceidentity can employ the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.), using default parametersprovided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotides orpolypeptides over a “comparison window” to identify and compare localregions of sequence similarity. In some embodiments, a “referencesequence” can be based on a primary amino acid sequence, where thereference sequence is a sequence that can have one or more changes inthe primary sequence. For instance, a “reference sequence based on SEQID NO:4 having at the residue corresponding to X14 a valine” or X14Vrefers to a reference sequence in which the corresponding residue at X14in SEQ ID NO:4, which is a tyrosine, has been changed to valine.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent identity and 89 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 residue positions, frequentlyover a window of at least 30-50 residues, wherein the percentage ofsequence identity is calculated by comparing the reference sequence to asequence that includes deletions or additions which total 20 percent orless of the reference sequence over the window of comparison. Inspecific embodiments applied to polypeptides, the term “substantialidentity” means that two polypeptide sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 80 percent sequence identity, preferably at least 89 percentsequence identity, at least 95 percent sequence identity or more (e.g.,99 percent sequence identity). Preferably, residue positions which arenot identical differ by conservative amino acid substitutions.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredimine reductase, can be aligned to a reference sequence by introducinggaps to optimize residue matches between the two sequences. In thesecases, although the gaps are present, the numbering of the residue inthe given amino acid or polynucleotide sequence is made with respect tothe reference sequence to which it has been aligned.

“Amino acid difference” or “residue difference” refers to a change inthe amino acid residue at a position of a polypeptide sequence relativeto the amino acid residue at a corresponding position in a referencesequence. The positions of amino acid differences generally are referredto herein as “Xn,” where n refers to the corresponding position in thereference sequence upon which the residue difference is based. Forexample, a “residue difference at position X25 as compared to SEQ IDNO:2” refers to a change of the amino acid residue at the polypeptideposition corresponding to position 25 of SEQ ID NO:2. Thus, if thereference polypeptide of SEQ ID NO:2 has a valine at position 25, then a“residue difference at position X25 as compared to SEQ ID NO:2” an aminoacid substitution of any residue other than valine at the position ofthe polypeptide corresponding to position 25 of SEQ ID NO:2. In mostinstances herein, the specific amino acid residue difference at aposition is indicated as “XnY” where “Xn” specified the correspondingposition as described above, and “Y” is the single letter identifier ofthe amino acid found in the engineered polypeptide (i.e., the differentresidue than in the reference polypeptide). In some embodiments, theremore than one amino acid can appear in a specified residue position, thealternative amino acids can be listed in the form XnY/Z, where Y and Zrepresent alternate amino acid residues. In some instances (e.g., inTables 3A, 3B, 3C, 3D and 3E), the present invention also providesspecific amino acid differences denoted by the conventional notation“AnB”, where A is the single letter identifier of the residue in thereference sequence, “n” is the number of the residue position in thereference sequence, and B is the single letter identifier of the residuesubstitution in the sequence of the engineered polypeptide. Furthermore,in some instances, a polypeptide of the present invention can includeone or more amino acid residue differences relative to a referencesequence, which is indicated by a list of the specified positions wherechanges are made relative to the reference sequence. The presentinvention includes engineered polypeptide sequences comprising one ormore amino acid differences that include either/or both conservative andnon-conservative amino acid substitutions.

“Conservative amino acid substitution” refers to a substitution of aresidue with a different residue having a similar side chain, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. By way of example and not limitation, an amino acid with analiphatic side chain may be substituted with another aliphatic aminoacid, e.g., alanine, valine, leucine, and isoleucine; an amino acid withhydroxyl side chain is substituted with another amino acid with ahydroxyl side chain, e.g., serine and threonine; an amino acid havingaromatic side chains is substituted with another amino acid having anaromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, andhistidine; an amino acid with a basic side chain is substituted withanother amino acid with a basic side chain, e.g., lysine and arginine;an amino acid with an acidic side chain is substituted with anotheramino acid with an acidic side chain, e.g., aspartic acid or glutamicacid; and a hydrophobic or hydrophilic amino acid is replaced withanother hydrophobic or hydrophilic amino acid, respectively. Exemplaryconservative substitutions are provided in Table 1 below.

TABLE 1 Residue Possible Conservative Substitutions A, L, V, I Otheraliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Othernon-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic(K, R) N, Q, S, T Other polar H, Y, W, F Other aromatic (H, Y, W, F) C,P None

“Non-conservative substitution” refers to substitution of an amino acidin the polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine), (b) the charge or hydrophobicity, or (c) the bulkof the side chain. By way of example and not limitation, an exemplarynon-conservative substitution can be an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the reference enzymewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered imine reductase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. In some embodiments,the improved engineered imine reductase enzymes comprise insertions ofone or more amino acids to the naturally occurring polypeptide havingimine reductase activity as well as insertions of one or more aminoacids to other improved imine reductase polypeptides. Insertions can bein the internal portions of the polypeptide, or to the carboxy or aminoterminus. Insertions as used herein include fusion proteins as is knownin the art. The insertion can be a contiguous segment of amino acids orseparated by one or more of the amino acids in the naturally occurringpolypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of the full-length imine reductasepolypeptide, for example the polypeptide of any of SEQ ID NOS:4-1300.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The engineered imine reductase enzymes may be present withina cell, present in the cellular medium, or prepared in various forms,such as lysates or isolated preparations. As such, in some embodiments,the engineered imine reductase enzyme can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure imine reductase compositionwill comprise about 60% or more, about 70% or more, about 80% or more,about 90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species. In some embodiments, the isolatedengineered imine reductase polypeptide is a substantially purepolypeptide composition.

“Stereoselective” refers to a preference for formation of onestereoisomer over another in a chemical or enzymatic reaction.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” refers to a chemical or enzymatic reaction thatis capable of converting a substrate or substrates, e.g., substratecompounds (1e) and (2b), to the corresponding amine product, e.g.,compound (3i), with at least about 85% stereomeric excess.

“Improved enzyme property” refers to an imine reductase polypeptide thatexhibits an improvement in any enzyme property as compared to areference imine reductase. For the engineered imine reductasepolypeptides described herein, the comparison is generally made to thewild-type enzyme from which the imine reductase is derived, although insome embodiments, the reference enzyme can be another improvedengineered imine reductase. Enzyme properties for which improvement isdesirable include, but are not limited to, enzymatic activity (which canbe expressed in terms of percent conversion of the substrate), thermostability, solvent stability, pH activity profile, cofactorrequirements, refractoriness to inhibitors (e.g., substrate or productinhibition), stereospecificity, and stereoselectivity (includingenantioselectivity).

“Increased enzymatic activity” refers to an improved property of theengineered imine reductase polypeptides, which can be represented by anincrease in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount of iminereductase) as compared to the reference imine reductase enzyme.Exemplary methods to determine enzyme activity are provided in theExamples. Any property relating to enzyme activity may be affected,including the classical enzyme properties of K_(m), V_(max) or k_(cat),changes of which can lead to increased enzymatic activity. Improvementsin enzyme activity can be from about 1.2 times the enzymatic activity ofthe corresponding wild-type enzyme, to as much as 2 times, 5 times, 10times, 20 times, 25 times, 50 times or more enzymatic activity than thenaturally occurring or another engineered imine reductase from which theimine reductase polypeptides were derived Imine reductase activity canbe measured by any one of standard assays, such as by monitoring changesin properties of substrates, cofactors, or products. In someembodiments, the amount of products generated can be measured by LiquidChromatography-Mass Spectrometry (LC-MS). Comparisons of enzymeactivities are made using a defined preparation of enzyme, a definedassay under a set condition, and one or more defined substrates, asfurther described in detail herein. Generally, when lysates arecompared, the numbers of cells and the amount of protein assayed aredetermined as well as use of identical expression systems and identicalhost cells to minimize variations in amount of enzyme produced by thehost cells and present in the lysates.

“Conversion” refers to the enzymatic conversion of the substrate(s) tothe corresponding product(s). “Percent conversion” refers to the percentof the substrate that is converted to the product within a period oftime under specified conditions. Thus, the “enzymatic activity” or“activity” of a imine reductase polypeptide can be expressed as “percentconversion” of the substrate to the product.

“Thermostable” refers to a imine reductase polypeptide that maintainssimilar activity (more than 60% to 80% for example) after exposure toelevated temperatures (e.g., 40-80° C.) for a period of time (e.g.,0.5-24 hrs) compared to the wild-type enzyme.

“Solvent stable” refers to an imine reductase polypeptide that maintainssimilar activity (more than e.g., 60% to 80%) after exposure to varyingconcentrations (e.g., 5-99%) of solvent (ethanol, isopropyl alcohol,dimethylsulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran,acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for aperiod of time (e.g., 0.5-24 hrs) compared to the wild-type enzyme.

“Thermo- and solvent stable” refers to an imine reductase polypeptidethat is both thermostable and solvent stable.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(m)) of the hybrids. In general, the stability of a hybrid is afunction of ion strength, temperature, G/C content, and the presence ofchaotropic agents. The T_(m) values for polynucleotides can becalculated using known methods for predicting melting temperatures (see,e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al.,1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc.Natl. Acad. Sci USA 83:8893-8897; Freier et al., 1986, Proc. Natl. Acad.Sci USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Rychliket al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991, NucleicAcids Res 19:698); Sambrook et al., supra); Suggs et al., 1981, InDevelopmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit Rev Biochem Mol Biol26:227-259. All publications incorporate herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered imine reductase enzyme of the present invention.

“Hybridization stringency” relates to hybridization conditions, such aswashing conditions, in the hybridization of nucleic acids. Generally,hybridization reactions are performed under conditions of lowerstringency, followed by washes of varying but higher stringency. Theterm “moderately stringent hybridization” refers to conditions thatpermit target-DNA to bind a complementary nucleic acid that has about60% identity, preferably about 75% identity, about 85% identity to thetarget DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding theimine reductase enzymes may be codon optimized for optimal productionfrom the host organism selected for expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (See, GCG CodonPreference, Genetics Computer Group WisconsinPackage; Codon W, John Peden, University of Nottingham; McInerney,Bioinform., 14:372-73 [1998]; Stenico et al., Nucl. Acids Res.,222437-46 [1994]; and Wright, Gene 87:23-29 [1990]). Codon usage tablesare available for a growing list of organisms (See e.g., Wada et al.,Nucl. Acids Res. 20:2111-2118 [1992]; Nakamura et al., Nucl. Acids Res.28:292 [2000]; Duret, et al., supra; and Henaut and Danchin, in“Escherichia coli and Salmonella,” Neidhardt et al. (Eds.), ASM Press,Washington D.C., [1996], p. 2047-2066). The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (See e.g., Mount, Bioinformatics:Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., [2001]; Uberbacher, Meth. Enzymol.,266:259-281 [1996]; and Tiwari et al., Comput. Appl. Biosci., 13:263-270[1997]).

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polynucleotideand/or polypeptide of the present invention. Each control sequence maybe native or foreign to the nucleic acid sequence encoding thepolypeptide. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest, such as acoding sequence. The promoter sequence contains transcriptional controlsequences, which mediate the expression of a polynucleotide of interest.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

“Suitable reaction conditions” refer to those conditions in thebiocatalytic reaction solution (e.g., ranges of enzyme loading,substrate loading, cofactor loading, temperature, pH, buffers,co-solvents, etc.) under which an imine reductase polypeptide of thepresent invention is capable of converting a substrate compound to aproduct compound (e.g., conversion of compound (2) to compound (1)).Exemplary “suitable reaction conditions” are provided in the presentinvention and illustrated by the Examples.

“Cofactor regeneration system” or “cofactor recycling system” refers toa set of reactants that participate in a reaction that reduces theoxidized form of the cofactor (e.g., NADP⁺ to NADPH). Cofactors oxidizedby the imine reductase catalyzed reductive amination of the ketonesubstrate are regenerated in reduced form by the cofactor regenerationsystem. Cofactor regeneration systems comprise a stoichiometricreductant that is a source of reducing hydrogen equivalents and iscapable of reducing the oxidized form of the cofactor. The cofactorregeneration system may further comprise a catalyst, for example anenzyme catalyst that catalyzes the reduction of the oxidized form of thecofactor by the reductant. Cofactor regeneration systems to regenerateNADH or NADPH from NAD⁺ or NADP⁺, respectively, are known in the art andmay be used in the methods described herein.

“Formate dehydrogenase” and “FDH” are used interchangeably herein torefer to an NAD or NADP⁺-dependent enzyme that catalyzes the conversionof formate and NAD⁺ or NADP⁺ to carbon dioxide and NADH or NADPH,respectively.

“Loading”, such as in “compound loading” or “enzyme loading” or“cofactor loading” refers to the concentration or amount of a componentin a reaction mixture at the start of the reaction.

“Substrate” in the context of a biocatalyst mediated process refers tothe compound or molecule acted on by the biocatalyst. For example, animine reductase biocatalyst used in the reductive amination processesdisclosed herein there is a ketone (or aldehyde) substrate of formula(I), such as cyclohexanone, and an amine substrate of formula (II), suchas butylamine.

“Product” in the context of a biocatalyst mediated process refers to thecompound or molecule resulting from the action of the biocatalyst. Forexample, an exemplary product for an imine reductase biocatalyst used ina process disclosed herein is a secondary or tertiary amine compound,such as a compound of formula (III).

“Alkyl” refers to saturated hydrocarbon groups of from 1 to 18 carbonatoms inclusively, either straight chained or branched, more preferablyfrom 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbonatoms inclusively. An alkyl with a specified number of carbon atoms isdenoted in parenthesis, e.g., (C₁-C₆)alkyl refers to an alkyl of 1 to 6carbon atoms.

“Alkylene” refers to a straight or branched chain divalent hydrocarbonradical having from 1 to 18 carbon atoms inclusively, more preferablyfrom 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbonatoms inclusively.

“Alkenyl” refers to groups of from 2 to 12 carbon atoms inclusively,either straight or branched containing at least one double bond butoptionally containing more than one double bond.

“Alkenylene” refers to a straight or branched chain divalent hydrocarbonradical having 2 to 12 carbon atoms inclusively and one or morecarbon-carbon double bonds, more preferably from 2 to 8 carbon atomsinclusively, and most preferably 2 to 6 carbon atoms inclusively.

“Alkynyl” refers to groups of from 2 to 12 carbon atoms inclusively,either straight or branched containing at least one triple bond butoptionally containing more than one triple bond, and additionallyoptionally containing one or more double bonded moieties.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 12 carbon atomsinclusively having a single cyclic ring or multiple condensed ringswhich can be optionally substituted with from 1 to 3 alkyl groups.Exemplary cycloalkyl groups include, but are not limited to, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl,1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and thelike, or multiple ring structures, including bridged ring systems, suchas adamantyl, and the like.

“Cycloalkylalkyl” refers to an alkyl substituted with a cycloalkyl,i.e., cycloalkyl-alkyl- groups, preferably having from 1 to 6 carbonatoms inclusively in the alkyl moiety and from 3 to 12 carbon atomsinclusively in the cycloalkyl moiety. Such cycloalkylalkyl groups areexemplified by cyclopropylmethyl, cyclohexylethyl and the like.

“Aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to12 carbon atoms inclusively having a single ring (e.g., phenyl) ormultiple condensed rings (e.g., naphthyl or anthryl). Exemplary arylsinclude phenyl, pyridyl, naphthyl and the like.

“Arylalkyl” refers to an alkyl substituted with an aryl, i.e.,aryl-alkyl- groups, preferably having from 1 to 6 carbon atomsinclusively in the alkyl moiety and from 6 to 12 carbon atomsinclusively in the aryl moiety. Such arylalkyl groups are exemplified bybenzyl, phenethyl and the like.

“Heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” refer to alkyl,alkenyl and alkynyl as defined herein in which one or more of the carbonatoms are each independently replaced with the same or differentheteroatoms or heteroatomic groups. Heteroatoms and/or heteroatomicgroups which can replace the carbon atoms include, but are not limitedto, —O—, —S—, —S—O—, —NR^(γ)—, —PH—, —S(O)—, —S(O)₂—, —S(O)NR^(γ)—,—S(O)₂NR^(γ)—, and the like, including combinations thereof, where eachR^(γ) is independently selected from hydrogen, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

“Heteroaryl” refers to an aromatic heterocyclic group of from 1 to 10carbon atoms inclusively and 1 to 4 heteroatoms inclusively selectedfrom oxygen, nitrogen and sulfur within the ring. Such heteroaryl groupscan have a single ring (e.g., pyridyl or furyl) or multiple condensedrings (e.g., indolizinyl or benzothienyl).

“Heteroarylalkyl” refers to an alkyl substituted with a heteroaryl,i.e., heteroaryl-alkyl- groups, preferably having from 1 to 6 carbonatoms inclusively in the alkyl moiety and from 5 to 12 ring atomsinclusively in the heteroaryl moiety. Such heteroarylalkyl groups areexemplified by pyridylmethyl and the like.

“Heterocycle”, “heterocyclic” and interchangeably “heterocycloalkyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, from 2 to 10 carbon ring atoms inclusively andfrom 1 to 4 hetero ring atoms inclusively selected from nitrogen, sulfuror oxygen within the ring. Such heterocyclic groups can have a singlering (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings(e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Examples ofheterocycles include, but are not limited to, furan, thiophene,thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, isothiazole, phenazine,isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline,piperidine, piperazine, pyrrolidine, indoline and the like.

“Heterocycloalkylalkyl” refers to an alkyl substituted with aheterocycloalkyl, i.e., heterocycloalkyl-alkyl- groups, preferablyhaving from 1 to 6 carbon atoms inclusively in the alkyl moiety and from3 to 12 ring atoms inclusively in the heterocycloalkyl moiety.

“Oxy” refers to a divalent group —O—, which may have varioussubstituents to form different oxy groups, including ethers and esters.

“Alkoxy” or “alkyloxy” are used interchangeably herein to refer to thegroup —OR^(ζ), wherein R^(ζ) is an alkyl group, including optionallysubstituted alkyl groups.

“Aryloxy” as used herein refer to the group —OR wherein R is an arylgroup as defined above including optionally substituted aryl groups asalso defined herein.

“Carboxy” refers to —COOH.

“Carboxyalkyl” refers to an alkyl substituted with a carboxy group.

“Carbonyl” refers to the group —C(O)—. Substituted carbonyl refers tothe group R^(η)—C(O)—R^(η), where each R^(η) is independently selectedfrom optionally substituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy,carboxy, aryl, aryloxy, heteroaryl, heteroarylalkyl, acyl,alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Typicalsubstituted carbonyl groups including acids, ketones, aldehydes, amides,esters, acyl halides, thioesters, and the like.

“Amino” refers to the group —NH₂. Substituted amino refers to the group—NHR^(η), NW^(η)R^(η), and NW^(η)R^(η)R^(η), where each R^(η) isindependently selected from optionally substituted alkyl, cycloalkyl,cycloheteroalkyl, alkoxy, carboxy, aryl, aryloxy, heteroaryl,heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, andthe like. Typical amino groups include, but are limited to,dimethylamino, diethylamino, trimethylammonium, triethylammonium,methylysulfonylamino, furanyl-oxy-sulfamino, and the like.

“Aminoalkyl” refers to an alkyl group in which one or more of thehydrogen atoms are replaced with an amino group, including a substitutedamino group.

“Aminocarbonyl” refers to a carbonyl group substituted with an aminogroup, including a substituted amino group, as defined herein, andincludes amides.

“Aminocarbonylalkyl” refers to an alkyl substituted with anaminocarbonyl group, as defined herein.

“Halogen” or “halo” refers to fluoro, chloro, bromo and iodo.

“Haloalkyl” refers to an alkyl group in which one or more of thehydrogen atoms are replaced with a halogen. Thus, the term “haloalkyl”is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. upto perhaloalkyls. For example, the expression “(C₁ C₂) haloalkyl”includes 1-fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl,1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1 trifluoroethyl,perfluoroethyl, etc.

“Hydroxy” refers to —OH.

“Hydroxyalkyl” refers to an alkyl substituted with one or more hydroxygroup.

“Thio” or “sulfanyl” refers to —SH. Substituted thio or sulfanyl refersto —S—R^(η), where R^(η) is an alkyl, aryl or other suitablesubstituent.

“Alkylthio” refers to —SR^(ζ), where R^(ζ) is an alkyl, which can beoptionally substituted. Typical alkylthio group include, but are notlimited to, methylthio, ethylthio, n-propylthio, and the like.

“Alkylthioalkyl” refers to an alkyl substituted with an alkylthio group,—SR^(ζ), where R^(ζ) is an alkyl, which can be optionally substituted.

“Sulfonyl” refers to —SO₂—. Substituted sulfonyl refers to —SO₂—R^(η),where R^(η) is an alkyl, aryl or other suitable substituent.

“Alkylsulfonyl” refers to —SO₂—R^(ζ), where R^(ζ) is an alkyl, which canbe optionally substituted. Typical alkylsulfonyl groups include, but arenot limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and thelike.

“Alkylsulfonylalkyl” refers to an alkyl substituted with analkylsulfonyl group, —SO₂—R^(ζ), where R^(ζ) is an alkyl, which can beoptionally substituted.

“Membered ring” is meant to embrace any cyclic structure. The numberpreceding the term “membered” denotes the number of skeletal atoms thatconstitute the ring. Thus, for example, cyclohexyl, pyridine, pyran andthiopyran are 6-membered rings and cyclopentyl, pyrrole, furan, andthiophene are 5-membered rings.

“Fused bicyclic ring” refers to both unsubstituted and substitutedcarbocyclic and/or heterocyclic ring moieties having 5 or 8 atoms ineach ring, the rings having 2 common atoms.

“Optionally substituted” as used herein with respect to the foregoingchemical groups means that positions of the chemical group occupied byhydrogen can be substituted with another atom, such as carbon, oxygen,nitrogen, or sulfur, or a chemical group, exemplified by, but notlimited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substitutedalkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo,methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substitutedalkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio,alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substitutedcarboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino,sulfonamido, substituted sulfonamido, cyano, amino, substituted amino,alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo,hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl,arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substitutedheteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl,heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl,cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle,(heterocycle)oxy, and (heterocycle)alkyl; where preferred heteroatomsare oxygen, nitrogen, and sulfur. Additionally, where open valencesexist on these substitute chemical groups they can be furthersubstituted with alkyl, cycloalkyl, aryl, heteroaryl, and/or heterocyclegroups, that where these open valences exist on carbon they can befurther substituted by halogen and by oxygen-, nitrogen-, orsulfur-bonded substituents, and where multiple such open valences exist,these groups can be joined to form a ring, either by direct formation ofa bond or by formation of bonds to a new heteroatom, preferably oxygen,nitrogen, or sulfur. It is further contemplated that the abovesubstitutions can be made provided that replacing the hydrogen with thesubstituent does not introduce unacceptable instability to the moleculesof the present invention, and is otherwise chemically reasonable. One ofordinary skill in the art would understand that with respect to anychemical group described as optionally substituted, only stericallypractical and/or synthetically feasible chemical groups are meant to beincluded. Finally, “optionally substituted” as used herein refers to allsubsequent modifiers in a term or series of chemical groups. Forexample, in the term “optionally substituted arylalkyl,” the “alkyl”portion and the “aryl” portion of the molecule may or may not besubstituted, and for the series “optionally substituted alkyl,cycloalkyl, aryl and heteroaryl,” the alkyl, cycloalkyl, aryl, andheteroaryl groups, independently of the others, may or may not besubstituted.

6.3 Engineered Imine Reductase (IRED) Polypeptides

The present invention provides engineered polypeptides having iminereductase activity, polynucleotides encoding the polypeptides; methodsof preparing the polypeptides, and methods for using the polypeptides.Where the description relates to polypeptides, it is to be understoodthat it also describes the polynucleotides encoding the polypeptides.

As noted above, imine reductases belong to a class of enzymes thatcatalyze the reductive amination of a ketone substrate and a primary orsecondary amine substrate to a secondary or tertiary amine product, asillustrated by Scheme 1 (see above for Scheme and group structures forcompounds of formula (I), (II), and (III)).

The opine dehydrogenase from Arthrobacter sp. strain 1C (also referredto herein as “CENDH”) having the amino acid sequence of SEQ ID NO:2,naturally catalyzes the conversion of ketone substrate, pyruvate and theamino acid substrate, L-2-amino pentanoic acid (or “L-norvaline”) to theproduct (2S)-2-((1-carboxyethyl)amino)pentanoic acid. CENDH alsocatalyzes the reaction of pyruvate with the amino acid substrates,L-ornithine, and β-alanine, and structurally similar amino sulfonic acidsubstrate, taurine. In addition, CENDH was found to catalyze theconversion of the unactivated ketone substrate, cyclohexanone (ratherthan pyruvate) and its natural amine substrate, L-norvaline, to thesecondary amine product, (S)-2-(cyclohexylamino)pentanoic acid. CENDHalso was found to catalyze the conversion of its natural ketonesubstrate pyruvate with the primary amines butylamine, ethylamine, andisopropylamine, to their respective 2-(alkylamino)propanoic acidsecondary amine products. CENDH, however, did not exhibit any activityfor the conversion of pyruvate with secondary amines, such asdimethylamine. Furthermore, CENDH did not show any imine reductaseactivity with the unactivated ketone substrate, cyclohexanone, when usedtogether with the unactivated primary amine substrate, butylamine.

The present invention provides engineered imine reductases that overcomethe deficiencies of the wild-type opine dehydrogenase CENDH. Theengineered imine reductase polypeptides derived from the wild-typeenzyme of Arthrobacter sp. strain 1C are capable of efficientlyconverting pyruvate and L-norvaline to the product(2S)-2-((1-carboxyethyl)amino)pentanoic acid, but also capable ofefficiently converting a range of ketone substrate compounds of formula(I) and amine substrate compounds of formula (II), to the secondary andtertiary amine product compounds of formula (III) as shown by conversionreactions (a) through (g) which are listed below in Table 2.

TABLE 2 Conversion Reactions Conversion Substrate Compound of SubstrateCompound Product Compound(s) of Reaction ID Formula (I) of Formula (II)Formula (III) (A)

(B)

(C)

(D)

(E)

(F)

(G)

Significantly, the present invention provides amino acid residuepositions and corresponding mutations in the sequence of the referenceengineered polypeptide having imine reductase activity of SEQ ID NOS: 4,6 or 8 (which was previously evolved from the naturally occurring CENDHpolypeptide of SEQ ID NO:2) that result in improved enzyme properties,including among others, imine reductase activity, substrate specificityand selectivity. In particular, the present invention providesengineered IRED polypeptides capable of catalyzing reductive aminationreactions such as those of Table 2 (i.e., the reductive amination ofketone substrate compounds of formula (I) with primary and secondaryamine substrate compounds of formula (II) thereby producing secondary ortertiary amine compounds of formula (III)).

In some embodiments, the engineered imine reductase polypeptides show anincreased activity in the conversion of the ketone substrate of formula(I) and amine substrate of formula (II) to an amine product of formula(III), in a defined time with the same amount of enzyme as compared tothe wild-type enzyme, CENDH. In some embodiments, the engineered iminereductase polypeptide has at least about 1.2 fold, 1.5 fold, 2 fold, 3fold, 4 fold, 5 fold, or 10 fold or more the activity as compared to thereference engineered polypeptide of SEQ ID NOS:4, 6 or 8, under suitablereaction conditions.

In some embodiments, the engineered imine reductase polypeptides exhibitan imine reductase activity in the conversion of a ketone substrate offormula (I) and an amine substrate of formula (II) to an amine productof formula (III), for which the wild-type polypeptide of SEQ ID NO:2,CENDH, has no detectable activity.

The product compounds of formula (III) produced by the engineered iminereductase polypeptides can be a secondary or tertiary amine compoundshaving one or more chiral centers. In some embodiments, the engineeredimine reductase polypeptides are capable of converting the ketone andamine substrate compounds of formula (I) and formula (II), to a chiralamine product compound of formula (III), in an enantiomeric excess ordiastereomeric excess of greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or greater.

In some embodiments, the engineered imine reductase polypeptides arecapable of converting the ketone and amine substrate compounds offormula (I) and formula (II) with increased tolerance for the presenceof one or both of these substrate compounds relative to the tolerance ofthe reference polypeptide of SEQ ID NOS:4, 6 or 8, under suitablereaction conditions. Thus, in some embodiments the engineered iminereductase polypeptides are capable of converting the ketone and aminesubstrate compounds of formula (I) and formula (II) at a substrateloading concentration of at least about 1 g/L, about 5 g/L, about 10g/L, about 20 g/L, about 30 g/L, about 40 g/L, about 50 g/L, about 70g/L, about 100 g/L, about 125 g/L, about 150 g/L. about 175 g/L or about200 g/L or more with a percent conversion of at least about 0.001%, atleast about 0.01%, at least about 0.1%, at least about 1%, at leastabout 10%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 98%, or at least about 99%, in a reaction timeof about 120 h or less, 72 h or less, about 48 h or less, about 36 h orless, or about 24 h less, under suitable reaction conditions.

The suitable reaction conditions under which the above-describedimproved properties of the engineered polypeptides carry out theconversion can be determined with respect to concentrations or amountsof polypeptide, substrate, cofactor (e.g., NAD(P)H), coenzyme (e.g., FDH[formate dehydrogenase] or GDH [glucose dehydrogenase]), buffer,co-solvent, pH, temperature, reaction time, and/or conditions with thepolypeptide immobilized on a solid support, as further described belowand in the Examples.

The present invention provides numerous exemplary engineeredpolypeptides having imine reductase activity. These exemplarypolypeptides were evolved from the previously engineered polypeptide ofSEQ ID NOS:4, 6 or 8 (which were obtained via directed evolution fromthe wild-type CENDH of SEQ ID NO:2) and exhibit improved properties,particularly increased activity and stability in the conversion ofvarious ketone and amine substrates, including the conversion ofcompounds (1a) and (2a) to the amine product compound (3a), theconversion of compounds (1b) and (2b) to the amine product compound(3b), the conversion of compounds (1c) and (2c) to the amine productcompound (3c), the conversion of compounds (1d) and (2d) to the amineproduct compound (3d), the conversion of compounds (1d) and (2c) to theamine product compound (3e), the conversion of compounds (1d) and (2c)to the amine product compound (3f), and the conversion of compounds (1d)and (2c) to the amine product compound (3g). These exemplary engineeredpolypeptides having imine reductase activity have amino acid sequences(provided in the accompanying Sequence Listing as even-numbered sequenceidentifiers of SEQ ID NOS:10-1300) that include one or more residuedifferences as compared to SEQ ID NOS: 4, 6 or 8 at the followingresidue positions: X9, X12, X31, X37, X44, X46, X48, X55, X56, X65, X82,X93, X98, X108, X137, X138, X141, X142, X143, X153, X154, X155, X156,X159, X166, X167, X168, X173, X177, X178, X184, X185, X195, X197, X198,X199, X200, X201, X203, X205, X206, X210, X213, X215, X216, X218, X220,X221, X223, X224, X226, X234, X239, X242, X245, X251, X253, X256, X257,X259, X260, X261, X262, X263, X265, X266, X267, X268, X269, X272, X273,X274, X276, X277, X278, X279, X280, X281, X282, X283, X284, X285, X289,X290, X291, X292, X293, X294, X330, and X349. The results in Tables3A-3M provide the variants with increased imine reductase activityand/or stability across a range of ketone and amine substrates.

The structure and function information for exemplary non-naturallyoccurring (or engineered) imine reductase polypeptides of the presentinvention are based on seven different high-throughput (HTP) screeningassays used in the directed evolution of these enzymes: the conversionof compounds (1a) and (2a) to the amine product compound (3a), theconversion of compounds (1b) and (2b) to the amine product compound(3b), the conversion of compounds (1c) and (2c) to the amine productcompound (3c), the conversion of compounds (1d) and (2d) to the amineproduct compound (3d), the conversion of compounds (1d) and (2c) to theamine product compound (3e), the conversion of compounds (1d) and (2c)to the amine product compound (3f), and the conversion of compounds (1d)and (2c) to the amine product compound (3g). The results of these HTPscreening assays which are shown below in Tables 3A-3M. The odd numberedsequence identifiers (i.e., SEQ ID NOs) refer to the nucleotide sequenceencoding the amino acid sequence provided by the even numbered SEQ IDNOs, and the sequences are provided in the electronic sequence listingfile accompanying this invention, which is hereby incorporated byreference herein.

TABLE 3A Results for Transformation A Increased Activity Relative SEQ IDto SEQ ID NO: 4 NO: Amino Acid Differences Transformation (A) 44° C.,(nt/aa) (Relative to SEQ ID NO: 4) pH 8.0, 15% DMSO  9/10 E198D +++11/12 Y153G +++ 13/14 Y153Q ++ 15/16 Y153N + 17/18 Y153R + 19/20A167T/Q265T + 21/22 Y153E + 23/24 S262V + Legend for Table 3A +++ >= 2.5++ >= 1.8 + >= 1.2

TABLE 3B Increased Activity Relative to SEQ ID NO: 12 SEQ IDTransformation (A) NO: Amino Acid Differences 44° C., pH 8.0, 15%(nt/aa) (Relative to SEQ ID NO: 4) DMSO 25/26N108C/Y153G/E198D/A234C/S284Q ++++ 27/28 N108C/Y153G/E198D/A234C/G282L++++ 29/30 N108C/Y153G/E198D/H220Q/A234C/E272D/G282L/S284Q ++++ 31/32N108C/Y153G/E198D/A234C ++++ 33/34 N108C/Y153G/E198D/S284Q ++++ 35/36N108C/Y153G/E198D/A234C ++++ 37/38 N108C/Y153G/E198D/A234C/E256S ++++39/40 N108C/Y153G/E198D/A234C/P267A ++++ 41/42N108C/Y153G/E198D/A234C/G282L ++++ 43/44 N108C/Y153G/E198D/H220Q ++++45/46 N108C/Y153G/E198D/H220Q/A234C ++++ 47/48N108C/Y153G/E198D/A234C/E256S ++++ 49/50 N108C/Y153G/E198D/A234C/S284C++++ 51/52 N108C/Y153G/E198D/H220Q/A234C/I242C ++++ 53/54N108C/Y153G/E198D/H220Q/A234C/S284H ++++ 55/56N108C/Y153G/E198D/H220Q/A234C/E272D/G282L/S284L ++++ 57/58N108C/Y153G/E198D/H220Q/S284L +++ 59/60 N108C/Y153G/E198D/H220Q/I242C+++ 61/62 Y153G/E198D/A234C/S284Q +++ 63/64N108C/Y153G/E198D/A234C/I242C/E256S/S284L +++ 65/66N108C/Y153G/E198D/H220Q/A234C/E256S/P267A +++ 67/68N108C/Y153G/E198D/H220Q/A234C/I242C/E256S +++ 69/70Y153G/E198D/A234C/E256S/G282L +++ 71/72N108C/Y153G/E198D/A234C/E256S/R281L +++ 73/74 Y153G/E198D/E272D +++75/76 N108C/Y153G/I197M/E198D/H220Q/A234C/S284L +++ 77/78N108C/Y153G/I197M/E198D/A234C/G282L +++ 79/80Y153G/E198D/H220Q/A234C/I242C/P267A/E272D +++ 81/82N108C/Y153G/I197M/E198D/A234C +++ 83/84 Y153G/E198D/A234C/I242C/S284L+++ 85/86 N108C/Y153G/I197M/E198D/A234C +++ 87/88N108C/Y153G/I197M/E198D/H220Q/A234C/E256S/E272D/G282L/ +++ S284H 89/90N108C/Y153G/I197M/H220Q/A234C/G282L/S284H +++ 91/92N108C/Y153G/I197M/E198D/H220Q/P267A/E272D/S284Q ++ 93/94Y153G/E198D/H220Q/I242C ++ 95/96N108C/Y153G/I197M/E198D/H220Q/A234C/I242C ++ 97/98N108C/Y153G/I197M/A234C/E256S/Q265V ++  99/100 Y153G/E198D/A234C/E256V++ 101/102 N108C/Y153G/I197M/E198D/H220Q/A234C/E272D ++ 103/104N108C/Y153G/I197M/A234C/G282L/S284H ++ 105/106N108C/Y153G/E198D/E256A/Q265V/G282L/S284C ++ 107/108N108C/Y153G/I197M/E198D/A234C/E256S ++ 109/110N108C/Y153G/I197M/H220Q/A234C/E256S/G282L ++ 111/112N108C/Y153G/I197M/E256S/S262V/E272D ++ 113/114 N108C/Y153G/I197M/A234C++ Legend for Table 3B ++++ >= 5 +++ >= 2.5 ++ >= 1.8 + >= 1.2

TABLE 3C Increased Activity Relative to SEQ SEQ ID NO: 30 ID NO: AminoAcid Differences Transformation (A) (nt/aa) (Relative to SEQ ID NO: 4)44° C., pH 8.0, 15% DMSO 115/116 N108C/Y153G/E198D/H220Q/ + A234C/E272D/R281N/G282L/S284Q 117/118 N108C/Y153G/E198D/H220Q/ + A234C/E272D/R281P/G282L/S284Q 119/120 N108C/Y153G/E198D/H220Q/ + A234C/E272D/L277A/G282L/S284Q 121/122 N108C/Y153G/E198D/H220Q/ + E224D/A234C/E272D/G282L/S284Q 123/124 N108C/R143K/Y153G/E198D/ + H220Q/A234C/E272D/G282L/S284Q Legend for Table 3C +++ >= 2.5 ++ >= 1.8 + >= 1.2

TABLE 3D SEQ Increased Activity Relative to SEQ ID NO: Amino AcidDifferences ID NO: 6 Transformation (B) (nt/aa) (Relative to SEQ ID NO:6) 44° C., pH 8.0, 15% DMSO 125/126 V283T +++ 127/128 S262F +++ 129/130V283W +++ 131/132 V9G/V259M +++ 133/134 H220E ++ 135/136 V283E ++137/138 P267S ++ 139/140 S262N ++ 141/142 Y153L ++ 143/144 Y153L +145/146 V279A + 147/148 V279Y + 149/150 P267Y + 151/152 V200S + 153/154Y153M + 155/156 V279H + 157/158 Y153M + 159/160 V259I + 161/162 P267N +163/164 P267I + 165/166 E224H + 167/168 E256W + Legend for Table 3D+++ >= 2.5 ++ >= 1.8 + >= 1.2

TABLE 3E Increased Activity Relative to SEQ ID NO: 126 SEQ IDTransformation (B) NO: Amino Acid Differences 44° C., (nt/aa) (Relativeto SEQ ID NO: 6) pH 8.0, 15% DMSO 169/170 N137D/R143K/P267Y/V279A/V283T+++ 171/172 N137D/H220E/V279H/V283T +++ 173/174N137D/S262F/P267Y/V279H/V283T +++ 175/176 V200S/S262F/P267Y/V279Y/V283T++ 177/178 P267S/V279H/V283T ++ 179/180 S262F/V279Y/V283T ++ 181/182P267S/V279Y/V283T ++ 183/184 P267T/V279Y/V283T ++ 185/186V259M/K260R/I261R/V283T ++ 187/188 N137D/E256C/P267Y/V283T ++ 189/190S262N/V279Y/V283T ++ 191/192 N137D/V200S/P267Y/V283T ++ 193/194N137D/E256C/V279Y/V283T ++ 195/196 P267S/V283T ++ 197/198N137D/P267S/V283T ++ 199/200 N137D/P267S/V279A/V283T ++ 201/202V259I/I261R/V283T ++ 203/204 N137D/P267Y/V283T ++ 205/206Y153L/I261R/V283T ++ 207/208 P267S/V279A/V283T ++ 209/210N137D/P267Y/V279A/V283T ++ 211/212 N137D/V279H/V283T + 213/214V200S/P267Y/V283T + 215/216 N137D/E256C/V283T + 217/218N137D/F154V/V283T + 219/220 N137D/P267Y/V279H/V283T + 221/222N137D/P267S/V279N/V283T + 223/224 N137D/V279Y/V283T + 225/226E256C/P267Y/V283T + 227/228 S262N/V283T + 229/230V200S/S262F/P267Y/V283T + 231/232 N137D/E256W/P267S/V279H/V283T +233/234 N137D/H220E/V279N/V283T + 235/236 N137D/V279A/V283T + 237/238E256W/P267S/V283T + 239/240 S262N/P267S/V279N/V283T + 241/242V279H/V283T + 243/244 N137D/Y153M/V279A/V283T + 245/246P267N/V279Y/V283T + 247/248 K260R/I261R/V283T + 249/250N137D/E256C/G276S/V283T + 251/252 N137D/V279L/V283T + 253/254E256W/P267Y/V279Y/V283T + 255/256 P267S/V279N/V283T + 257/258V259I/I261R/V283W + 259/260 N137D/V283T + 261/262 I261R/V283T + 263/264Y153L/V283T + 265/266 N137D/A185T/V279A/V283T + 267/268 H220E/V283T +Legend for Table 3E +++ >= 2.5 ++ >= 1.8 + >= 1.2

TABLE 3F SEQ Increased Activity Relative to SEQ ID NO: Amino AcidDifferences ID NO: 174 Transformation (B) (nt/aa) (Relative to SEQ IDNO: 6) 44° C., pH 8.0, 15% DMSO 269/270N137D/R143K/Y153L/H220E/S262F/P267Y/V279H/ +++ V283T 271/272N137D/R143K/H220E/E256C/S262F/P267Y/V279H/ ++ V283T 273/274N137D/R143K/V200S/H220E/V259M/S262F/P267Y/ ++ V279H/V283T 275/276N137D/R143K/H220E/V259M/S262F/P267Y/V279H/ ++ V283T 277/278N137D/R143K/V200S/E256C/V259M/S262F/P267S/ ++ V279H/V283T 279/280N137D/R143K/Y153L/V200S/H220E/E256C/I261R/ ++ S262F/P267S/V279H/V283T281/282 N137D/R143K/V200S/H220E/E256C/V259M/I261R/ ++S262F/P267Y/V279H/V283T 283/284N137D/R143K/V200S/H220E/E256C/S262F/P267Y/ ++ V279H/V283T 285/286N137D/R143K/Y153L/E256C/V259M/S262F/P267Y/ ++ V279H/V283T 287/288N137D/R143K/E256C/V259I/S262F/P267Y/V279H/ ++ V283T 289/290N137D/R143K/F154V/H220E/S262F/P267S/V279H/ ++ V283T 291/292N137D/R143K/Y153L/F154V/H220E/S262F/P267S/ ++ V279H/V283T 293/294N137D/R143K/Y153L/F154V/H220E/S262F/P267Y/ ++ V279H/V283T 295/296N137D/R143K/F154V/H220E/E256C/V259M/I261R/ ++ S262F/P267S/V279H/V283T297/298 N137D/Y153L/H220E/S262F/P267Y/V279H/V283T ++ 299/300N137D/R143K/V259I/S262F/P267Y/V279H/V283T ++ 301/302N137D/R143K/F154V/S262F/P267Y/V279H/V283T ++ 303/304N137D/R143K/V200S/H220E/E256C/V259I/I261R/ ++ S262F/P267Y/V279H/V283T305/306 N137D/R143K/E256C/V259M/S262F/P267Y/V279H/ ++ V283T 307/308N137D/R143K/H220E/V259I/I261R/S262F/P267Y/ ++ V279H/V283T 309/310N137D/R143K/Y153L/V200S/H220E/V259M/S262F/ ++ P267Y/V279H/V283T 311/312N137D/R143K/H220E/S262F/P267Y/V279H/V283T ++ 313/314N137D/R143K/V200S/H220E/V259M/I261R/S262F/ ++ P267Y/V279H/V283T 315/316N137D/R143K/E256C/V259M/S262F/P267S/V279H/ ++ V283T 317/318N137D/R143K/H220E/E256C/S262F/P267S/V279H/ ++ V283T 319/320N137D/R143K/Y153L/H220E/S262F/P267S/V279H/ ++ V283T 321/322N137D/R143K/F154V/H220E/S262F/P267Y/V279H/ ++ V283T 323/324N137D/R143K/H220E/S262F/P267S/V279H/V283T ++ 325/326N137D/R143K/H220E/V259M/I261R/S262F/P267Y/ ++ V279H/V283T 327/328N137D/R143K/V200S/H220E/E256C/I261R/S262F/ + P267Y/V279H/V283T 329/330N137D/R143K/V200S/E256C/S262F/P267Y/V279H/ + V283T 331/332N137D/R143K/S262F/P267Y/V279H/V283T + 333/334N137D/R143K/H220E/E256C/I261R/S262F/P267Y/ + V279H/V283T 335/336N137D/R143K/S262F/P267S/V279H/V283T + 337/338N137D/R143K/Y153L/F154V/S262F/P267S/V279H/ + V283T 339/340N137D/R143K/E256C/S262F/P267Y/V279H/V283T + 341/342N137D/Y153L/H220E/E256C/I261R/S262F/P267S/ + V279H/V283T 343/344N137D/R143K/H220E/V259I/S262F/P267S/V279H/ + V283T 345/346N137D/R143K/Y153L/S262F/P267S/V279H/V283T + 347/348N137D/R143K/E256C/V259M/I261R/S262F/P267Y/ + V279H/V283T 349/350N137D/R143K/V259M/S262F/P267Y/V279H/V283T + 351/352N137D/R143K/V259M/I261R/S262F/P267S/V279H/ + V283T 353/354N137D/R143K/V200S/E256C/V259I/I261R/S262F/ + P267Y/V279H/V283T 355/356N137D/H220E/E256C/I261R/S262F/P267Y/V279H/ + V283T 357/358N137D/H220E/S262F/P267Y/V279H/V283T + Legend for Table 3F +++ >= 2.5++ >= 1.8 + >= 1.2

TABLE 3G Increased Increased Increased Activity Relative ActivityRelative Activity Relative to SEQ ID NO: 8 to SEQ ID NO: 8 to SEQ ID NO:8 SEQ ID Transformation Transformation Transformation NO: Amino AcidDifferences (C) 44° C., pH 8.0, (D) 44° C., pH 8.0, (E) 44° C., pH 8.0,(nt/aa) (Relative to SEQ ID NO: 8) 15% DMSO 15% DMSO 15% DMSO 359/360V283W ++++ + 361/362 V283Y +++ 363/364 M12L/V65L/I201L/C234A/ +++ ++++N260K/H261I/C263Q/V283T/ E292V 365/366 M12L/A37P/V65L/I201L/C234A/ +++++++ H261I/V262S/V283T/ E292V 367/368 M12L/A37P/V65L/I201L/C234A/ +++++++ V283T/E292V 369/370 M12L/V65L/I201L/C234A/ +++ +A256E/N260K/C263Q/L280P/ E292V 371/372 M12L/V65L/Q159M/C234A/ ++ A256E373/374 L173M/L289W ++ ++ + 375/376 M12L/T223S/C234A/R281A ++ ++ 377/378I155M ++ + 379/380 M12L/V65L/T156M/Q159M/ ++ C234A/N260D/S284P 381/382Y153A ++ 383/384 M138L ++ +++ 385/386 T156E + 387/388M12L/V65L/T223S/C234A + + 389/390 C263T/G282T + 391/392 I155S + +393/394 P278V + 395/396 Q265N + + 397/398 Y153G + + 399/400 R46S + +401/402 V65L/Y153G + + 403/404 L289R + ++ + 405/406 G55Y + ++ + 407/408T206N + 409/410 M12L/V65L/T156M/Q159M/ + C234A/N260D/C263E/S284P 411/412G177T + + 413/414 F154M + ++ 415/416 N290R + + 417/418 L56M + 419/420M12L/I201L/C234A/V262S/ ++++ V283T/E292V 421/422 G55C + 423/424 P267Q +425/426 N290S + 427/428 N290R/L349P + 429/430 N137D +++ 431/432 C213M+++ 433/434 A273V ++ 435/436 T206V ++ 437/438 R281S ++ 439/440 V279R ++441/442 T206L ++ 443/444 T206D ++ 445/446 V65F ++ 447/448 T82V ++449/450 Q159E ++ 451/452 P267V ++ 453/454 A273I ++ 455/456 A256S ++457/458 Q184L/G216V ++ 459/460 V279E ++ 461/462 A273T + 463/464 I226Y +465/466 E257S + 467/468 C213L + 469/470 T206H + 471/472 Y221W + 473/474T206I + 475/476 I242V + 477/478 P218Q + 479/480 S178E + 481/482 A239Q +483/484 A48G + 485/486 A210F + 487/488 Y221L + 489/490 T269H + 491/492L31M/T195S + 493/494 C213Y + 495/496 G177A + 497/498 A185G + 499/500A268N + 501/502 N251E + 503/504 G285W + 505/506 L280E + 507/508 T206Q +509/510 R143L + 511/512 R46N + 513/514 P253D + 515/516 F294A + 517/518Q44N + 519/520 C213F + 521/522 K168M + 523/524 A166I + 525/526 F154V +527/528 F154L + 529/530 S215E + 531/532 A185L + 533/534 I242C + 535/536I330V + 537/538 A93G + 539/540 C234M + 541/542 T266N + 543/544 Y98W +Legend for Table 3G ++++ >= 5.0 +++ >= 2.5 ++ >= 1.8 + >= 1.2

TABLE 3H Increased Activity Increased Activity Relative to SEQ IDRelative to SEQ ID SEQ ID NO: 360 Transformation NO: 360 TransformationNO: Amino Acid Differences (F) 44° C., pH 8.0, 15% (G) 44° C., pH 8.0,15% (nt/aa) (Relative to SEQ ID NO: 8) DMSO DMSO 545/546W141T/I242V/V283W ++++ 547/548 W141V/C142A/M274V/V283W/ ++++ E292V549/550 W141V/C142A/V283W/S284M/ ++++ E292V 551/552 W141T/V283W ++++553/554 W141V/R143W/Y153E/V283W/ +++ E292V 555/556W141T/Y153F/V259L/M274V/V283W/ +++ E292V 557/558 W141V/Y153F/V283W +++559/560 W141T/I242V/E257S/V283W +++ 561/562 W141T/Q184L/A256S/V283W +++563/564 W141T/Q184L/V283W +++ 565/566 W141T/A256S/V283W +++ 567/568W141V/I201L/V259M/V283W +++ 569/570 V65F/N137D/T206V/V283W ++++ +++571/572 T206V/P267V/V283W +++ +++ 573/574 W141V/Y153E/V283W ++ 575/576W141T/Q184L/I242V/V283W ++ 577/578 V65F/P267V/V279R/V283W +++ ++ 579/580V65F/Q159E ++ ++ 581/582 W141T/C142A/V283W/S284H ++ 583/584 N137D/V283W++++ ++ 585/586 W141-/I201L/V259L/V283W ++ 587/588 T206L/P267V/R281S ++++ 589/590 W141T/C142A/V283W ++ 591/592 T82V/N137D/T206V/V279R/R281S++++ ++ 593/594 T206L/R281S/V283W ++++ ++ 595/596 I242V/V283W/G285W + ++597/598 V65F/Q159E/V279R ++ ++ 599/600 V65F/N137D/V283W ++++ ++ 601/602N137D/V279R/V283W +++ ++ 603/604 V65F/N137D +++ ++ 605/606 V279E/V283W++++ ++ 607/608 W141T/C142A/R143W/I201L/V259L/ ++ V283W 609/610C213Y/I242V/E257S/V283W ++ 611/612 V65F/T82V/N137D +++ ++ 613/614Q159E/V283W +++ + 615/616 V279E ++ + 617/618V65F/N137D/P267V/V279R/V283W +++ + 619/620 V65F/V279R/R281S; V283W++++ + 621/622 V283W ++ + 623/624 V65F/P267V/V279R/R281S/V283W ++++ +625/626 W141T/R143W/Y153E/V283W/S284H + 627/628 W141T/V279E + 629/630G216V/P218Q/I226Y/I242V/A256S/ + V279E 631/632 N137D/T206V/V283W +++ +633/634 T82V/Q159E/V279R/R281S/V283W ++++ + 635/636W141T/C142A/R143W/V259M/ + M274V/V283W 637/638 V65F/R281S/V283W +++ +639/640 W141T/V279E/G285W + 641/642 N137D/V279R/R281S/V283W ++++ +643/644 I226Y/I242V/V283W + 645/646 A256S + 647/648 I242V/V279E/V283W++++ + 649/650 E257S + 651/652 V65F/T82V/T206L/A273I/V283W +++ + 653/654Q159E/V279R ++ + 655/656 V65F/T206L/A273V/R281S/V283W ++++ + 657/658N137D/A273V/V283W ++++ + 659/660 V65F/V279R/V283W ++++ + 661/662V65F/T82V/N137D/A273V/R281S ++++ + 663/664 C213L/Y221L/E257S/V283W/G285W+++ + 665/666 V65F/N137D/T206V/R281S/V283W ++++ + 667/668C213M/R281S/V283W +++ + 669/670 V279E/V283W/G285W ++ + 671/672T206L/V279R/R281S/V283W ++++ + 673/674 N137D/P267V/V283W +++ + 675/676T82V/T206L/R281S/V283W ++++ + 677/678 T82V/T206L/A273I/V283W +++ +679/680 R46S/G55Y/F154M/L173M/T206N/ ++ + L289W 681/682V65F/N137D/P267V/V279R/R281S +++ + 683/684F154M/I155M/L173M/T206N/Q265N/ +++ + G282T/V283W 685/686R46S/L173M/T206N/G282T/V283W/ +++ + L349P 687/688R46S/F154M/I155M/T206N/G282T/ +++ + V283W 689/690 R46S/T206N/V283W +++ +691/692 Y153F/I201L/V259M/M274V/R281A/ ++++ + V283W/S284H/E292V 693/694C213M/A273V/V283W +++ + 695/696 G55C/F154M/T206N/C263T/G282T/ ++++ +V283W 697/698 G282T/V283W ++ + 699/700 T82V/R281S/V283W ++++ + 701/702C213L/V279E/V283W/G285W ++ + 703/704 V65F/N137D/C213M/V283W +++ +705/706 N137D/R281S/V283W ++++ + 707/708 T82V/Q159E/T206L/V279R/V283W+++ + 709/710 I242V/A256S/V279E/V283W/G285W ++ + 711/712L173M/T206N/G282T/V283W/L349P +++ + 713/714 N137D/P267V/R281S/V283W+++ + 715/716 R46S/I155M/T206N/V283W +++ + 717/718I226Y/I242V/V279E/V283W +++ + 719/720 R46S/G55C/I155M/L173M/T206N/ +++ +Q265N/G282T/V283W 721/722 V65F/N137D/A273V/V283W ++++ + 723/724V65F/N137D/R281S/V283W ++++ + 725/726 L173M/T206N/G282T/V283W ++++727/728 R46S/I155M/L173M/T206N/G282T/ +++ V283W 729/730R46S/L173M/T206N/V283W/L289W ++ 731/732 R46S/G55C/L173M/T206N/Q265N/ +++G282T/V283W/L349P 733/734 N137D/T206L/R281S/V283W ++++ 735/736G55C/T206N/V283W ++ 737/738 V279R/V283W ++++ 739/740 R46S/G282T/V283W ++741/742 N137D/T206L/V283W +++ 743/744 V65F/N137D/T206L/V283W ++++745/746 E257S/V279E/V283W ++++ 747/748 V279R/R281S/V283W ++++ 749/750T206N/G282T/V283W/L349P +++ 751/752 R46S/L173M/T206N/V283W +++ 753/754N137D/Q159E/V279R/V283W +++ 755/756 Q159E/V279R/R281S/V283W ++++ 757/758N137D/C213M/V283W +++ 759/760 R46S/G55Y/T206N/V283W/L349P +++ 761/762V65F/T206D/V279R/R281S/V283W +++ 763/764 T82V/T206L/V279R/R281S ++++765/766 E257S/V279E/V283W/G285W ++ 767/768 T82V/N137D/T206L/P267V/V279R/+++ R281S 769/770 N137D/T206V/R281S ++ 771/772G55C/T206N/C263T/G282T/V283W/ +++ L349P 773/774 L173M/T206N/V283W +++775/776 T206L/V283W ++ 777/778 T82V/Q159E/T206L/R281S/V283W ++++ 779/780A273V/V279R/R281S +++ 781/782 R46S/T206N/G282T/V283W/N290R ++++ 783/784R46S/L173M/T206N/G282T/V283W/ ++++ N290R 785/786R46S/T206N/C263T/G282T/V283W ++++ 787/788 G55C/T206N/Q265N/G282T/V283W++++ 789/790 P267V/V279R/V283W ++ 791/792 V283W/E292V ++ 793/794R46S/G55Y/I155M/L173M/T206N/ +++ G282T/V283W/L349P 795/796R46S/G55C/T206N/G282T/V283W ++++ 797/798 V65F/N137D/T206L/P267V/R281S/+++ V283W 799/800 L173M/T206N/G282T/V283W/N290R ++++ 801/802N137D/V279R/R281S +++ 803/804 R46S/L173M/T206N/Q265N/G282T/ ++++ V283W805/806 T82V/Q159E/C213M/V279R +++ 807/808 Q159E/V279R/V283W +++ 809/810R46S/T206N/V283W/L349P +++ 811/812 T82V/N137D/V283W ++++ 813/814T82V/N137D +++ 815/816 R46S/G55C/L173M/T206N/G282T/ +++ V283W/L349P817/818 R46S/L173M/T206N/G282T/V283W ++++ 819/820 R46S/T206N/G282T/V283W++++ 821/822 Q184L/A256S/V279E/V283W ++++ 823/824N137D/P267V/V279R/R281S +++ 825/826 I242V/V279E/V283W/G285W ++ 827/828T206N/G282T/V283W/L289W +++ 829/830 R46S/G55C/I155M/L173M/T206N/ +++G282T/V283W 831/832 T206N/G282T/V283W ++++ 833/834V65F/T206L/V279R/R281S/V283W ++++ 835/836 Q184L/I242V/V279E/G285W ++837/838 V65F/T82V/N137D/P267V/V279R/ ++ R281S/V283W 839/840Q159E/A273V/V279R/V283W ++ Legend for Table 3H ++++ >= 5.0 +++ >= 2.5++ >= 1.8 + >= 1.2

TABLE 3I Increased Activity Increased Activity Relative to SEQ IDRelative to SEQ ID SEQ ID NO: 550 Transformation NO: 550 TransformationNO: Amino Acid Differences (F) 44° C., pH 8.0, 15% (G) 44° C., pH 8.0,15% (nt/aa) (Relative to SEQ ID NO: 8) DMSO DMSO 841/842W141V/C142A/P205G/V283W/ ++++ S284M/E292V 843/844W141V/C142A/P205G/V283W/ ++++ S284M/E292V 845/846W141V/C142A/Y153E/P205D/ ++++ V283W/S284M/E292V 847/848W141V/C142A/Y153E/P205A/ ++++ V283W/S284M/E292V 849/850W141V/C142A/Y153E/P205A/ ++++ V283W/S284M/E292V 851/852W141V/C142A/P205T/V283W/ ++++ S284M/E292V 853/854W141V/C142A/Y153E/P205G/ ++++ V283W/S284M/E292V 855/856W141V/C142A/Y153E/P205N/ +++ V283W/S284M/E292V 857/858W141V/C142A/Y153E/P205L/ + +++ V283W/S284M/E292V 859/860W141V/C142A/Y153E/P205L/ +++ V283W/S284M/E292V 861/862W141V/C142A/Y153E/P205L/ +++ V283W/S284M/E292V 863/864W141V/C142A/I197L/V283W/S284M/ +++ E292V 865/866L31M/A48G/W141V/C142A/T195S/ + +++ T206L/E257S/V279E/V283W/ S284M/E292V867/868 W141V/C142A/V283W/S284M/ +++ E292V/H293F 869/870W141V/C142A/Y153E/I201A/V283W/ +++ S284M/E292V 871/872W141S/C142A/V283W/S284M/ + +++ E292V 873/874 W141V/C142A/Q159L/V283W/ ++++ S284M/E292V 875/876 A93G/Y98W/W141V/C142A/Y153E/ + +++K168M/S178E/A268N/V283W/ S284M/E292V 877/878 W141S/C142A/V283W/S284M/+++ E292V 879/880 W141V/C142A/I201F/V283W/S284M/ + +++ E292V 881/882W141V/C142A/T156Q/V283W/ + +++ S284M/E292V 883/884L31M/V65F/W141V/C142A/T206L/ + +++ V279E/V283W/S284M/E292V 885/886W141S/C142A/V283W/S284M/ ++ E292V 887/888 A93G/Y98W/W141V/C142A/Y153E/++ K168M/A239Q/N251E/A268N/ V283W/S284M/E292V 889/890L31M/W141V/C142A/T206L/E257S/ + ++ V279E/V283W/S284M/E292V 891/892W141V/C142A/Q159L/V283W/ ++ S284M/E292V 893/894 W141V/C142A/I201C/V283W/++ S284M/E292V 895/896 W141V/C142A/T156Q/V283W/ ++ S284M/E292V 897/898W141V/C142A/Q159L/V283W/ ++ S284M/E292V 899/900 W141V/C142A/T206M/V283W/++ S284M/E292V 901/902 A93G/W141V/C142A/Y153E/K168M/ ++N251E/T269H/V283W/S284M/ E292V/I330V 903/904 W141V/C142A/K168M/A239Q/ ++N251E/A268N/V283W/S284M/ E292V/I330V 905/906Q44N/Y98W/W141V/C142A/Y153E/ + ++ K168M/S215E/A239Q/A268N/V283W/S284M/E292V 907/908 W141V/C142A/T156Q/V283W/ ++ S284M/E292V909/910 W141V/C142A/T156Q/V283W/ ++ S284M/E292V 911/912W141V/C142A/Y153E/V283W/ ++ S284M/N290Y/E292V 913/914W141V/C142A/Y153E/V283W/ ++ S284M/E292V/H293L 915/916W141V/C142A/P203G/V283W/ ++ S284M/E292V 917/918 W141S/C142A/V283W/S284M/++ E292V 919/920 L31M/W141V/C142A/T195S/V279E/ ++ V283W/S284M/E292V921/922 W141V/C142A/T156A/V283W/ ++ S284M/E292V 923/924Y98W/W141V/C142A/Y153E/ ++ K168M/A185G/A239Q/N251E/A268N/V283W/S284M/E292V/I330V 925/926 L31M/A48G/W141V/C142A/T195S/ ++E257S/V279E/V283W/S284M/ E292V 927/928 W141V/C142A/T206N/V283W/ ++S284M/E292V 929/930 L31M/A48G/W141V/C142A/T206L/ + ++V279E/V283W/S284M/E292V 931/932 L31M/A48G/V65L/W141V/C142A/ + ++T206L/V279E/V283W/S284M/ E292V 933/934 Q44N/W141V/C142A/V283W/S284M/ ++E292V 935/936 L31M/W141V/C142A/T206L/T266N/ + ++ V279E/V283W/S284M/E292V937/938 W141V/C142A/V283W/S284M/ ++ E292V/H293M 939/940A93G/Y98W/W141V/C142A/Y153E/ ++ K168M/N251E/V283W/S284M/ E292V/I330V941/942 A93G/Y98W/W141V/C142A/Y153E/ ++ K168M/A239Q/A268N/V283W/S284M/E292V 943/944 A93G/Y98W/W141V/C142A/Y153E/ ++K168M/A185G/S215E/N251E/ A268N/V283W/S284M/E292V 945/946W141V/C142A/K168M/A239Q/ ++ N251E/A268N/V283W/S284M/ E292V/I330V 947/948W141V/C142A/Y153E/K168M/ + ++ V283W/S284M/E292V 949/950W141V/C142A/Y153E/V200Y/ ++ L277M/V283W/S284M/E292V 951/952L31M/W141V/C142A/T206L/V279E/ + ++ V283W/S284M/E292V 953/954W141V/C142A/A256K/V283W/ ++ S284M/E292V 955/956L31M/W141V/C142A/T206L/E257S/ + ++ T266N/V283W/S284M/E292V 957/958W141V/C142A/I201F/V283W/S284M/ ++ E292V 959/960A93G/W141V/C142A/Y153E/K168M/ ++ S215E/A268N/V283W/S284M/ E292V/I330V961/962 W141V/C142A/Y153E/K168M/ ++ A239Q/A268N/V283W/S284M/ E292V963/964 W141V/C142A/T195R/V283W/ ++ S284M/E292V 965/966Y98W/W141V/C142A/Y153E/ ++ K168M/A185G/A239Q/N251E/A268N/V283W/S284M/E292V 967/968 R46N/W141V/C142A/Y153E/K168M/ ++A239Q/A268N/T269H/V283W/ S284M/E292V 969/970 W141V/C142A/H220D/V283W/ +++ S284M/E292V 971/972 W141V/C142A/M274V/V283W/ ++ S284M/E292V 973/974Q44N/Y98W/W141V/C142A/Y153E/ ++ K168M/A185G/S215E/A239Q/N251E/T269H/V283W/S284M/ E292V/I330V 975/976 W141V/C142A/V283W/S284M/ ++T291A/E292V 977/978 W141V/C142A/Y153E/V283W/ ++ S284I/E292V 979/980R46N/A93G/Y98W/W141V/C142A/ ++ Y153E/K168M/N251E/A268N/T269H/V283W/S284M/E292V/ I330V 981/982 W141V/C142A/P267K/V283W/ + ++S284M/E292V 983/984 Y98W/W141V/C142A/Y153E/ ++ K168M/S215E/A239Q/N251E/A268N/V283W/S284M/E292V/I330V 985/986 L31M/W141V/C142A/T206L/E257S/ + ++T266N/V279E/V283W/S284M/ E292V 987/988 W141V/C142A/A256N/V283W/ ++S284M/E292V 989/990 W141V/C142A/A256G/V283W/ ++ S284M/E292V 991/992R46N/A93G/Y98W/W141V/C142A/ ++ Y153E/K168M/A185L/A268N/T269H/V283W/S284M/E292V/ I330V 993/994 W141V/C142A/Y153E/I201M/ +V283W/S284M/E292V 995/996 W141V/C142A/Q159I/V283W/ + S284M/E292V 997/998A93G/Y98C/W141V/C142A/Y153E/ + K168M/A185G/N251E/A268D/T269H/V283W/S284M/E292V/ I330V  999/1000 A93G/Y98W/W141V/C142A/Y153E/ +K168M/A239Q/N251E/T269H/ V283W/S284M/E292V 1001/1002W141V/C142A/Y221I/V283W/ + S284M/E292V 1003/1004W141V/C142A/Y153E/I197L/V283W/ + S284M/E292V 1005/1006W141V/C142A/T156A/V283W/ + S284M/E292V 1007/1008L31M/A48G/V65L/W141V/C142A/ + + T206L/E257S/T266N/V279E/V283W/S284M/E292V 1009/1010 R46N/A93G/Y98W/W141V/C142A/ +Y153E/K168M/A239Q/A268N/ V283W/S284M/E292V/I330V 1011/1012L31M/A48G/W141V/C142A/T206L/ ++ + E257S/V279E/V283W/S284M/ E292V1013/1014 W141V/C142A/K168M/S215E/ ++ + A239Q/N251E/A268N/V283W/S284M/E292V/I330V 1015/1016 L31M/A48G/W141T/C142A/T206L/ +++ +E257S/V279E/V283W/S284M/ E292V 1017/1018 L31M/W141T/C142A/Y153E/T206L/++ T266N/V279E/V283W/S284M/E292V Legend for Table 3I ++++ >=5.0+++ >=2.5 ++ >=1.8 + >=1.2

TABLE 3J Increased Activity Relative to SEQ ID NO: 872 SEQ ID Amino AcidDifferences Transformation (G) 44° C., NO: (nt/aa) (Relative to SEQ IDNO: 8) pH 8.0, 15% DMSO 1019/1020W141V/C142A/T156G/P205T/T206N/H220D/A239Q/ +++++A256G/H261D/V283W/S284M/E292V/I330V 1021/1022W141S/C142A/P205T/T206N/H220D/A239Q/A256G/ +++++ V283W/S284M/E292V/H293Q1023/1024 W141S/C142A/T156A/P205T/T206N/H220D/A256G/ ++++H261D/V283W/S284M/E292V/H293Q 1025/1026W141S/C142A/P205T/A239Q/V279E/V283W/S284M/ ++++ E292V/H293F 1027/1028W141S/C142A/P205T/T206N/V283W/S284M/E292V/I330V ++++ 1029/1030W141S/C142A/T156Q/I201M/P205T/T206N/V283W/S284M/ ++++ E292V 1031/1032W141S/C142A/T156Q/P205T/H261D/V283W/S284M/ ++++ E292V/H293Q 1033/1034W141S/C142A/P205T/H220D/A239Q/A256G/V283W/ ++++ S284M/E292V 1035/1036W141S/C142A/I201M/P205T/T206N/A256G/H261E/V283W/ ++++ S284M/E292V1037/1038 W141S/C142A/P205T/A256G/V283W/S284M/E292V/ ++++ H293Q1039/1040 W141S/C142A/P205T/T206N/V283W/S284M/E292V/ ++++ H293Q1041/1042 W141S/C142A/P205T/A256G/V283W/S284M/E292V ++++ 1043/1044W141S/C142A/T156Q/P205T/V283W/S284M/E292V/ ++++ H293Q/I330V 1045/1046W141S/C142A/P205T/T206M/H261E/V283W/S284M/ ++++ E292V 1047/1048W141S/C142A/P205T/H220D/V283W/S284M/E292V/I330V ++++ 1049/1050W141S/C142A/P205T/T206M/A256G/V283W/S284M/ ++++ E292V/I330V 1051/1052W141S/C142A/P205T/H261E/V283W/S284M/E292V ++++ 1053/1054W141S/C142A/T156Q/P205T/T206M/H261D/V283W/ ++++ S284M/E292V 1055/1056M138L/W141V/C142A/P203G/P205T/V279F/V283W/ ++++ S284M/E292V 1057/1058W141S/C142A/P205T/A239Q/A256K/V283W/S284M/ ++++ E292V 1059/1060W141S/C142A/P205T/T206M/V283W/S284M/E292V ++++ 1061/1062W141S/C142A/K168M/P205T/T206M/V283W/S284M/ ++++ E292V 1063/1064W141S/C142A/P205T/A239Q/A256N/V283W/S284M/ ++++ E292V 1065/1066W141S/C142A/P205T/A256N/V283W/S284M/E292V ++++ 1067/1068W141S/C142A/P205T/V279E/V283W/S284M/E292V/ ++++ H293M 1069/1070W141S/C142A/P205T/V283W/S284M/E292V ++++ 1071/1072L31M/W141S/C142A/P205T/T206M/V279E/V283W/S284M/ ++++ E292V/I330V1073/1074 W141S/C142A/P205T/Y221I/A239Q/V283W/S284M/E292V ++++ 1075/1076W141S/C142A/P205T/V279E/V283W/S284M/E292V ++++ 1077/1078W141S/C142A/P205T/T206M/A245G/V279E/V283W/ ++++ S284M/E292V 1079/1080W141S/C142A/P205T/V283W/S284M/E292V/H293M ++++ 1081/1082N108S/W141S/C142A/K168M/I197L/C213V/A256N/V283W/ ++++ S284M/T291A/E292V1083/1084 L31M/V65L/W141V/C142A/P205T/V279E/V283W/S284M/ ++++E292V/H293F 1085/1086 W141S/C142A/P205T/C213V/A239Q/V283W/S284M/ ++++E292V/H293Y 1087/1088 W141V/C142A/P205S/A256G/V283W/S284M/E292V ++++1089/1090 W141V/C142A/T156Q/P205S/H220D/A239Q/A256G/ +++H261E/V283W/S284M/E292V/H293Q 1091/1092W141V/C142A/T156Q/P205S/H220D/A239Q/A256G/ +++H261E/V283W/S284M/E292V/I330V 1093/1094W141S/C142A/P205T/V283W/S284M/T291A/E292V +++ 1095/1096W141V/C142A/T156G/I201A/P205T/T206N/H220D/H261D/ +++V283W/S284M/E292V/H293Q/I330V 1097/1098W141V/C142A/I201A/P205T/T206N/H220D/H261D/V283W/ +++ S284M/E292V1099/1100 W141V/C142A/I201M/P205T/T206N/H220D/A239Q/ +++H261D/V283W/S284M/E292V 1101/1102W141S/C142A/K168M/P205T/A239Q/V283W/S284M/ +++ E292V 1103/1104W141S/C142A/P203G/P205T/V283W/S284M/E292V +++ 1105/1106W141S/C142A/K168M/P205T/V283W/S284M/E292V +++ 1107/1108W141S/C142A/P205T/C263Q/V279F/V283W/S284M/ +++ E292V 1109/1110W141V/C142A/T156A/P205S/H220D/A239Q/A256G/ +++ V283W/S284M/E292V/H293Q1111/1112 W141V/C142A/T156Q/P205S/H220D/H261D/V283W/ +++ S284M/E292V1113/1114 W141V/C142A/P205S/H220D/A239Q/A256G/H261D/ +++V283W/S284M/E292V/H293Q 1115/1116W141V/C142A/T156A/P205S/H220D/A256G/H261D/ +++ V283W/S284M/E292V1117/1118 W141V/C142A/T156A/P205S/H220D/V283W/S284M/ +++E292V/H293Q/I330V 1119/1120W141V/C142A/T156G/I201A/P205T/T206N/H220D/A256G/ +++H261E/V283W/S284M/E292V 1121/1122W141V/C142A/P205S/H220D/H261D/V283W/S284M/ +++ E292V/H293Q 1123/1124M138L/W141V/C142A/T156G/P205S/H220D/A239Q/ +++A256G/V283W/S284M/E292V/H293F/I330V 1125/1126W141V/C142A/P205S/H220D/A239Q/A256G/H261D/ +++ V283W/S284M/E292V1127/1128 W141V/C142A/T156G/P205S/H261E/V283W/S284M/ +++ E292V/H293Q1129/1130 W141V/C142A/P205S/H220D/H261E/V283W/S284M/ +++ E292V/H293Q1131/1132 W141V/C142A/P205S/H220D/H261D/V283W/S284M/ +++ E292V 1133/1134W141V/C142A/P205S/H220D/A239Q/H261E/V283W/ +++ S284M/E292V 1135/1136W141V/C142A/T156G/P205S/H220D/A239Q/A256G/ +++H261D/V283W/S284M/E292V/H293Q 1137/1138W141V/C142A/A199V/P205T/H220D/H261E/V283W/ +++ S284M/E292V/H293Q1139/1140 W141V/C142A/P205S/H220D/A239Q/A256G/H261E/ +++V283W/S284M/E292V/I330V 1141/1142W141V/C142A/P205T/T206N/H261E/V283W/S284M/ +++ E292V/I330V 1143/1144W141V/C142A/I197L/P205S/C263E/V79F/V283W/S284M/ +++ E292V 1145/1146W141V/C142A/I197L/P205S/V279F/V283W/S284M/E292V +++ 1147/1148M138L/W141V/C142A/P205S/A239Q/V279F/V283W/ +++ S284M/T291A/E292V1149/1150 M138L/W141V/C142A/P205S/V279F/V283W/S284M/ +++ T291A/E292V1151/1152 W141V/C142A/P205T/A239Q/V279F/V283W/S284M/ +++ E292V 1153/1154M138L/W141V/C142A/P205T/A239Q/V279F/V283W/ ++ S284M/E292V 1155/1156W141V/C142A/P205S/A239Q/V279F/V283W/S284M/ ++ E292V 1157/1158W141V/C142A/I197L/P205S/A239Q/V283W/S284M/E292V ++ 1159/1160W141V/C142A/I197L/P205S/V283W/S284M/E292V ++ 1161/1162W141V/C142A/P205S/A256K/V279F/V283W/S284M/ ++ E292V 1163/1164W141V/C142A/I197L/P205S/C213V/A256L/V283W/S284M/ ++ E292V 1165/1166M138L/W141V/C142A/I197L/P205S/V283W/S284M/ ++ E292V 1167/1168W141V/C142A/P205S/A239Q/A256L/V279F/V283W/ ++ S284M/E292V 1169/1170W141V/C142A/P205T/A256L/V279F/V283W/S284M/ ++ E292V 1171/1172W141V/C142A/K168M/I197L/P205S/Y221M/C263E/ ++M274V/V279F/V283W/S284M/E292V 1173/1174N108S/W141V/C142A/K168M/P205S/V279F/V283W/ ++ S284M/E292V 1175/1176M138L/W141V/C142A/P205S/V279F/V283W/S284M/ ++ E292V 1177/1178W141V/C142A/K168M/P205S/A256N/V279F/V283W/ + S284M/E292V 1179/1180W141V/C142A/P205S/V279F/V283W/S284M/E292V + 1181/1182W141V/C142A/K168M/I197L/P205S/A256L/V279F/V283W/ + S284M/E292V 1183/1184W141V/C142A/K168M/P205S/A239Q/V279F/V283W/ + S284M/E292V 1185/1186W141V/C142A/P203G/P205T/T266P/V279F/V283W/S284M/ + E292V Legend forTable 3J +++++ >=15.0 ++++ >=5.0 +++ >=2.5 ++ >=1.8 + >=1.2

TABLE 3K Increased Activity Relative SEQ ID to SEQ ID NO: 1020 NO: AminoAcid Differences Transformation (G) (nt/aa) (Relative to SEQ ID NO: 8)44° C., pH 8.0, 15% DMSO 1187/1188N108S/M138L/W141V/C142A/T156Q/K168M/I197L/ +P205T/T206N/H220D/A239Q/A256G/H261D/V279F/ V283W/S284M/E292V/H293Q/I330V1189/1190 N108S/M138L/W141V/C142A/T156Q/K168M/I197T/ +P205T/T206N/G216R/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/E292V/H293M/I330V 1191/1192N108S/W141V/C142A/T156G/K168M/A185L/I197T/ +P205T/T206N/H220D/A239Q/A256G/H261D/V279F/ V283W/S284M/E292V/H293M/I330V1193/1194 N108S/M138L/W141V/C142A/T156Q/K168M/I197L/ +P205T/T206N/G216R/H220D/A239Q/A256G/H261D/ V279F/V283W/S284M/E292V/I330V1195/1196 N108S/M138L/W141V/C142A/T156Q/K168M/I197L/ +P205T/T206N/G216S/H220D/A239Q/A256G/H261D/V279E/V283W/S284M/E292V/H293F/I330V 1197/1198N108S/M138L/W141V/C142A/T156A/K168M/A185L/ +P205T/T206N/H220D/A239Q/A256G/H261D/V279F/ V283W/S284M/E292V/H293F/I330VLegend for Table 3K +++++ >=15.0 ++++ >=5.0 +++ >=2.5 ++ >=1.8 + >=1.2

TABLE 3L Increased Activity Relative to SEQ ID NO: 1188 SEQ IDTransformation (G) NO: Amino Acid Differences 44° C., pH 8.0, 15%(nt/aa) (Relative to SEQ ID NO: 8) DMSO 1199/1200N14S/N108S/M138G/W141V/C142A/R143K/T156Q/K168M/I197L/ ++P205T/T206N/H220D/A239Q/A256G/H261D/V279F/V283W/ S284M/E292V/H293F/I330V1201/1202 A93T/N108S/M138G/W141V/C142A/R143K/T156Q/K168M/I197L/ ++P205T/T206N/H220D/A239Q/A256G/E257K/H261D/V279F/V283W/S284M/E292V/H293F/I330V 1203/1204N14S/A93T/N108S/N137D/M138L/W141V/C142A/R143K/T156Q/ ++K168M/I197L/P205T/T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/E292V/H293Q/I330V 1205/1206N108S/M138L/W141E/C142A/T156Q/K168M/I197L/P205T/T206N/ ++H220D/A239Q/A256G/H261D/V279F/V283W/S284M/E292V/ H293Q/I330V 1207/1208A93T/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ ++T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/ G285V/E292V/H293Q/I330V1209/1210 N108S/N137D/M138L/W141E/C142A/T156Q/K168M/I197L/P205T/ ++T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/ E292V/H293Q/I330V1211/1212 N108S/M138L/W141E/C142A/R143K/T156Q/K168M/I197L/P205T/ ++T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/ E292V/H293Q/I330V1213/1214 N108S/N137D/M138L/W141E/C142A/R143K/T156Q/K168M/ ++I197L/P205T/T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/E292V/H293Q/I330V 1215/1216N108S/M138L/W141V/C142A/T156Q/K168M/I197L/I201G/P205T/ ++T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/ E292V/H293Q/I330V1217/1218 N108S/N137D/M138L/W141V/C142A/T156Q/K168M/I197L/P205T/ ++T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/ E292V/H293Q/I330V1219/1220 N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/T206N/ ++H220D/A239Q/A256G/H261D/V279F/V283W/S284M/G285V/ E292V/H293Q/I330V1221/1222 N14S/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ ++T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/ E292V/H293F/I330V1223/1224 N108S/M138F/W141V/C142A/T156Q/K168M/I197L/P205T/T206N/ ++H220D/A239Q/A256G/H261D/V279F/V283W/S284M/E292V/ H293Q/I330V 1225/1226N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/T206N/ ++H220D/A239Q/A256G/H261D/V279F/V283W/S284M/E292V/ H293F/I330V 1227/1228N108S/N137D/M138L/W141V/C142A/T156Q/K168M/I197L/I201G/ +P205T/T206N/H220D/A239Q/A256G/H261D/V279F/V283W/ S284M/E292V/H293Q/I330V1229/1230 N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/T206N/ +H220D/A239Q/A256G/H261D/V279F/V283W/S284M/E292V/ H293Q/I330V 1231/1232N108S/M138G/W141V/C142A/R143K/T156Q/K168M/I197L/ +P205T/T206N/H220D/A239Q/A256G/H261D/V279F/V283W/ S284M/E292V/H293Q/I330V1233/1234 A93T/N108S/M138L/W141V/C142A/T156Q/K168M/I197L/P205T/ +T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/ E292V/H293F/I330V1235/1236 N108S/M138L/W141V/C142A/T156Q/K168M/I197L/P205T/T206N/ +H220D/A239Q/A256G/H261D/V279F/V283W/S284M/E292V/ H293F/I330V Legend forTable 3L ++ >=1.8 + >=1.2

TABLE 3M Increased Activity Relative to SEQ ID NO: 1220 SEQ IDTransformation (G) NO: Amino Acid Differences 44° C., pH 8.0, 15%(nt/aa) (Relative to SEQ ID NO: 8) DMSO 1237/1238W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ ++P205T/T206N/H220D/A239Q/A256G/V259I/H261D/V279F/V283W/S284M/G285V/E292V/H293F/I330V 1239/1240R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ ++T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/ G285V/E292V/H293F/I330V1241/1242 W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ ++P205T/T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/G285V/T287K/E292V/H293Q/S295K/I330V 1243/1244W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ ++P205T/T206N/H220D/A239Q/A256G/V259I/H261D/V279F/V283W/S284M/G285V/E292V/H293Q/S295K/I330V 1245/1246W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ ++P205T/T206N/H220D/A239Q/A256G/H261E/V279F/V283W/S284M/G285V/T287K/E292V/H293Q/I330V 1247/1248R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ ++T206N/H220D/A239Q/A256G/V259I/H261E/V279F/V283W/S284M/G285V/T287K/E292V/H293F/I330V 1249/1250R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ ++T206N/H220D/A239Q/A256G/V259I/H261E/G264S/V279F/V283W/S284M/G285V/E292V/H293F/I330V 1251/1252W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ ++P205T/T206N/H220D/A239Q/A256G/H261E/V279F/V283W/S284M/G285V/E292V/H293Q/S295K/I330V 1253/1254W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ ++P205T/T206N/H220D/A239Q/A256G/H261E/V279F/V283W/S284M/G285V/T287G/E292V/H293Q/I330V 1255/1256W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ ++P205T/T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/G285V/E292V/H293F/I330V 1257/1258W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ ++P205T/T206N/H220D/A239Q/A256G/V259I/H261D/V279F/V283W/S284M/G285V/T287G/E292V/H293Q/S295K/I330V 1259/1260R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ +T206N/H220D/A239Q/A256G/V259I/H261D/V279F/V283W/S284M/G285V/E292V/H293F/I330V/L341M 1261/1262W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ +P205T/T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/G285V/E292V/H293Q/S295K/I330V 1263/1264R39K/I43M/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ +P205T/T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/G285V/E292V/H293Q/S295K/I330V 1265/1266W33A/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ +T206N/H220D/A239Q/A256G/V259I/H261D/V279F/V283W/S284M/G285V/T287G/E292V/H293Q/S295K/I330V 1267/1268W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ +P205T/T206N/H220D/A239Q/A256G/V259I/H261D/V279F/V283W/S284M/G285V/E292V/H293Q/I330V 1269/1270W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ +P205T/T206N/H220D/A239Q/A256G/V259I/H261E/V279F/V283W/S284M/G285V/T287K/E292V/H293Q/I330V 1271/1272W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ +P205T/T206N/H220D/A239Q/A256G/V259I/H261E/V279F/V283W/S284M/G285V/E292V/H293Q/S295K/I330V 1273/1274R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ +T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/G285V/T287K/E292V/H293Q/I330V 1275/1276W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ +P205T/T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/G285V/E292V/H293Q/I330V 1277/1278W33A/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ +T206N/H220D/A239Q/A256G/V259I/H261E/V279F/V283W/S284M/G285V/E292V/H293Q/S295K/I330V 1279/1280W33A/R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/ +P205T/T206N/H220D/A239Q/A256G/V259I/H261E/V279F/V283W/S284M/G285V/T287G/E292V/H293Q/I330V 1281/1282W33A/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ +T206N/H220D/A239Q/A256G/V259I/H261D/V279F/V283W/S284M/G285V/T287K/E292V/H293Q/I330V 1283/1284R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ +T206N/H220D/A239Q/A256G/V259I/H261D/V279F/V283W/S284M/G285V/T287G/E292V/H293Q/I330V 1285/1286W33A/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ +T206N/H220D/A239Q/A256G/H261E/V279F/V283W/S284M/G285V/T287G/E292V/H293Q/I330V 1287/1288R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ +T206N/H220D/A239Q/A256G/H261E/V279F/V283W/S284M/G285V/E292V/H293Q/S295K/I330V 1289/1290R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ +T206N/H220D/A239Q/A256G/V259I/H261E/V279F/V283W/S284M/G285V/T287G/E292V/H293Q/I330V 1291/1292W33A/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ +T206N/H220D/A239Q/A256G/V259I/H261E/V279F/V283W/S284M/G285V/T287K/E292V/H293Q/I330V 1293/1294N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/T206N/ +H220D/A239Q/A256G/V259I/H261E/V279F/V283W/S284M/G285V/T287G/E292V/H293Q/S295K/I330V 1295/1296N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/T206N/ +H220D/A239Q/A256G/V259I/H261E/V279F/V283W/S284M/ G285V/E292V/H293F/I330V1297/1298 W33A/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ +T206N/H220D/A239Q/A256G/H261D/V279F/V283W/S284M/G285V/T287K/E292V/H293Q/I330V 1299/1300R39K/N108S/M138G/W141V/C142A/T156Q/K168M/I197L/P205T/ +T206N/H220D/A239Q/A256G/V259I/H261E/V279F/V283W/S284M/G285V/E292V/H293Q/I330V Legend for Table 3M 30 + >=1.8 + >=1.2

From an analysis of the exemplary polypeptides shown in Tables 3A-3M,improvements in enzyme properties are associated with residuedifferences as compared to the reference sequence of the engineeredpolypeptide of SEQ ID NO:4, 6, and/or 8, at the residue positionsindicated in these Tables.

Additionally, as noted above, the crystal structure of the opinedehydrogenase CENDH has been determined (See e.g., Britton et al., Nat.Struct. Biol., 5:593-601 [1998]). Accordingly, this correlation of thevarious amino acid differences and functional activity disclosed hereinalong with the known three-dimensional structure of the wild-type enzymeCENDH can provide the ordinary artisan with sufficient information torationally engineer further amino acid residue changes to thepolypeptides provided herein (and to homologous opine dehydrogenaseenzymes including OpDH, BADH, CEOS, and TauDH), and retain or improve onthe imine reductase activity or stability properties. In someembodiments, it is contemplated that such improvements can includeengineering the engineered polypeptides of the present invention to haveimine reductase activity with a range of substrates and provide a rangeof products as described in Scheme 1.

In light of the guidance provided herein, it is further contemplatedthat any of the exemplary engineered polypeptide sequences ofeven-numbered sequence identifiers SEQ ID NOS:4-1300, can be used as thestarting amino acid sequence for synthesizing other engineered iminereductase polypeptides, for example by subsequent rounds of evolution byadding new combinations of various amino acid differences from otherpolypeptides in Tables 3A-3M, and other residue positions describedherein. Further improvements may be generated by including amino aciddifferences at residue positions that had been maintained as unchangedthroughout earlier rounds of evolution. Accordingly, in someembodiments, the engineered polypeptide having imine reductase activitycomprises an amino acid sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the reference sequence of SEQ ID NO:2 and at leastone substitution as provided in Tables 3A-3M.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence comprising at least tworesidue differences as compared to the reference sequence of

As will be appreciated by the skilled artisan, in some embodiments, oneor a combination of residue differences above that is selected can bekept constant in the engineered imine reductases as a core sequence, andadditional residue differences at other residue positions incorporatedinto the core sequence to generate additional engineered imine reductasepolypeptides with improved properties. Accordingly, it is to beunderstood for any engineered imine reductase containing one or a subsetof the residue differences above, the present invention contemplatesother engineered imine reductases that comprise the one or subset of theresidue differences, and additionally one or more residue differences atthe other residue positions disclosed herein. By way of example and notlimitation, an engineered imine reductase comprising a residuedifference at residue position X9, can further incorporate one or moreresidue differences at the other residue positions (e.g., X198, etc.).

Generally, the engineered polypeptides having imine reductase activityof the present invention are capable of converting a compound of formula(I) and an compound of formula (II) to an amine product compound offormula (III) (as illustrated by Scheme 1) with improved activity and/orimproved stereoselectivity relative to the Arthrobacter sp. strain C1wild-type opine dehydrogenase reference polypeptide of SEQ ID NO:2, orrelative to a reference polypeptide having imine reductase activityselected from the engineered polypeptides of even-numbered sequenceidentifiers SEQ ID NOS:4-1300. In some embodiments, the improvedactivity and/or improved stereoselectivity is with respect to theconversion of a specific combination of a compound of formula (I) and acompound of formula (II) shown in Table 2 to the corresponding amineproduct compound of formula (III) shown in Table 2.

Accordingly, in some embodiments, the engineered polypeptides havingimine reductase activity of the present invention which have an aminoacid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a referencesequence selected from even-numbered sequence identifiers SEQ IDNOS:4-1300, and one or more residue differences as compared to SEQ IDNO:2 at residue positions selected from those provided herein, arecapable of one or more of the following conversion reactions, undersuitable reaction conditions, with improved activity and/or improvedstereoselectivity relative to a reference polypeptide of even-numberedsequence identifiers SEQ ID NOS:4-1300:

(a) conversion of substrate compounds (1a) and (2a) to product compound(3a);

(b) conversion of substrate compounds (1b) and (2b) to product compound(3b);

(c) conversion of substrate compounds (1c) and (2c) to product compound(3c);

(d) conversion of substrate compounds (1d) and (2d) to product compound(3d);

(e) conversion of substrate compounds (1d) and (2c) to product compound(3e);

(f) conversion of substrate compounds (1d) and (2c) to product compound(3f); and

(g) conversion of substrate compounds (1d) and (2c) to product compound(3g).

In some embodiments, the engineered polypeptide having imine reductaseactivity and capable of catalyzing one or more of the above conversionreactions (a)-(g), under suitable reaction conditions, with improvedactivity and/or stereoselectivity comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to one of even-numbered sequenceidentifiers SEQ ID NOS:4-1300, and the amino acid residue differences ascompared to SEQ ID NO:2 present in any one of even-numbered sequenceidentifiers SEQ ID NOS:4-1300, as provided in Tables 3A-3M.

In some embodiments, the engineered polypeptide having imine reductaseactivity and capable of catalyzing one or more of the above conversionreactions (a)-(g), under suitable reaction conditions, with improvedactivity and/or stereoselectivity has an amino acid sequence comprisinga sequence selected from the even-numbered sequence identifiers SEQ IDNOS:4-1300. The wild-type opine dehydrogenase from Arthrobacter sp.strain C1 (CENDH) of SEQ ID NO:2, from which the engineered polypeptidesof the present invention were derived has no detectable activity inconverting a ketone substrate of compound (1) and an amine substrate ofcompound (2) to a secondary amine product compound (3). In someembodiments, however, the engineered polypeptides having imine reductaseactivity are capable of converting the ketone substrates and aminesubstrates provided herein to a secondary amine product compound asdescribed herein.

Further, in some embodiments, the engineered polypeptide having iminereductase activity disclosed herein is capable of converting the ketonesubstrate of compound (1a) and the amine substrate of compound (2a) tothe amine product compound (3a) with at least 1.2 fold, 1.5 fold, 2fold, 3 fold, 4 fold, 5 fold, 10 fold or more activity relative to theactivity of the reference polypeptide of SEQ ID NO:4. In someembodiments, the engineered polypeptide having imine reductase activitydisclosed herein is capable of converting the ketone substrate ofcompound (1b) and the amine substrate of compound (2b) to the amineproduct compound (3b) with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold,4 fold, 5 fold, 10 fold or more activity relative to the activity of thereference polypeptide of SEQ ID NO:6. In some embodiments, theengineered polypeptide having imine reductase activity disclosed hereinis capable of converting the ketone substrate of compound (1c) and theamine substrate of compound (2c) to the amine product compound (3c) withat least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold ormore activity relative to the activity of the reference polypeptide ofSEQ ID NO:8. In some embodiments, the engineered polypeptide havingimine reductase activity disclosed herein is capable of converting theketone substrate of compound (1d) and the amine substrate of compound(2d) to the amine product compound (3d) with at least 1.2 fold, 1.5fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold or more activity relativeto the activity of the reference polypeptide of SEQ ID NO:8. In someembodiments, the engineered polypeptide having imine reductase activitydisclosed herein is capable of converting the ketone substrate ofcompound (1d) and the amine substrate of compound (2c) to the amineproduct compound (3e) with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold,4 fold, 5 fold, 10 fold or more activity relative to the activity of thereference polypeptide of SEQ ID NO:8. In some embodiments, theengineered polypeptide having imine reductase activity disclosed hereinis capable of converting the ketone substrate of compound (1d) and theamine substrate of compound (2c) to the amine product compound (3f) withat least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold ormore activity relative to the activity of the reference polypeptide ofSEQ ID NO: 8. In some embodiments, the engineered polypeptide havingimine reductase activity disclosed herein is capable of converting theketone substrate of compound (1d) and the amine substrate of compound(2c) to the amine product compound (3g) with at least 1.2 fold, 1.5fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold or more activity relativeto the activity of the reference polypeptide of SEQ ID NO: 8.

In addition to the positions of residue differences specified above, anyof the engineered imine reductase polypeptides disclosed herein canfurther comprise other residue differences at other residue positionsthan those of amino acid differences disclosed in Tables 3A-3M. Residuedifferences at these other residue positions can provide for additionalvariations in the amino acid sequence without adversely affecting theability of the polypeptide to catalyze one or more of the aboveconversion reactions (a)-(g) from Table 2. Accordingly, in someembodiments, in addition to the amino acid residue differences presentin any one of the engineered imine reductase polypeptides selected fromSEQ ID NOS:4-1300, the sequence can further comprise 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, or 1-50 residue differences atother amino acid residue positions as compared to the SEQ ID NO:2, 4, 6,and/or 8. In some embodiments, the number of amino acid residuedifferences as compared to the reference sequence can be 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 30, 30, 35, 40, 45 or 50 residue positions. In some embodiments, thenumber of amino acid residue differences as compared to the referencesequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,18, 20, 21, 22, 23, 24, or 25 residue positions. The residue differenceat these other positions can be conservative changes or non-conservativechanges. In some embodiments, the residue differences can compriseconservative substitutions and non-conservative substitutions ascompared to the naturally occurring imine reductase polypeptide of SEQID NO:2 or the engineered imine reductase polypeptide of SEQ ID NO:4, 6,and/or 8.

In some embodiments, the present invention also provides engineeredpolypeptides that comprise a fragment of any of the engineered iminereductase polypeptides described herein that retains the functionalactivity and/or improved property of that engineered imine reductase.Accordingly, in some embodiments, the present invention provides apolypeptide fragment capable of catalyzing one or more of the aboveconversion reactions (a)-(g) of Table 2, under suitable reactionconditions, wherein the fragment comprises at least about 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% of a full-length amino acid sequence of anengineered imine reductase polypeptide of the present invention, such asan exemplary engineered imine reductase polypeptide selected fromeven-numbered sequence identifiers SEQ ID NOS:4-1300.

In some embodiments, the engineered imine reductase polypeptide can havean amino acid sequence comprising a deletion of any one of theengineered imine reductase polypeptides described herein, such as theexemplary engineered polypeptides of even-numbered sequence identifiersSEQ ID NO:4-1300. Thus, for each and every embodiment of the engineeredimine reductase polypeptides of the invention, the amino acid sequencecan comprise deletions of one or more amino acids, 2 or more aminoacids, 3 or more amino acids, 4 or more amino acids, 5 or more aminoacids, 6 or more amino acids, 8 or more amino acids, 10 or more aminoacids, 15 or more amino acids, or 20 or more amino acids, up to 10% ofthe total number of amino acids, up to 10% of the total number of aminoacids, up to 20% of the total number of amino acids, or up to 30% of thetotal number of amino acids of the imine reductase polypeptides, wherethe associated functional activity and/or improved properties of theengineered imine reductase described herein is maintained. In someembodiments, the deletions can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35,1-40, 1-45, or 1-50 amino acid residues. In some embodiments, the numberof deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 aminoacid residues. In some embodiments, the deletions can comprise deletionsof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21,22, 23, 24, or 25 amino acid residues.

In some embodiments, the engineered imine reductase polypeptide hereincan have an amino acid sequence comprising an insertion as compared toany one of the engineered imine reductase polypeptides described herein,such as the exemplary engineered polypeptides of even-numbered sequenceidentifiers SEQ ID NOS:4-1300. Thus, for each and every embodiment ofthe imine reductase polypeptides of the invention, the insertions cancomprise one or more amino acids, 2 or more amino acids, 3 or more aminoacids, 4 or more amino acids, 5 or more amino acids, 6 or more aminoacids, 8 or more amino acids, 10 or more amino acids, 15 or more aminoacids, 20 or more amino acids, 30 or more amino acids, 40 or more aminoacids, or 50 or more amino acids, where the associated functionalactivity and/or improved properties of the engineered imine reductasedescribed herein is maintained. The insertions can be to amino orcarboxy terminus, or internal portions of the imine reductasepolypeptide.

In some embodiments, the engineered imine reductase polypeptide hereincan have an amino acid sequence comprising a sequence selected fromeven-numbered sequence identifiers SEQ ID NOS:4-1300, and optionally oneor several (e.g., up to 3, 4, 5, or up to 10) amino acid residuedeletions, insertions and/or substitutions. In some embodiments, theamino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40,1-45, or 1-50 amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the number of amino acid sequencehas optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residuedeletions, insertions and/or substitutions. In some embodiments, thesubstitutions can be conservative or non-conservative substitutions.

In the above embodiments, the suitable reaction conditions for theengineered polypeptides can be those HTP assay conditions described inTables 3A-3M and the Examples. Guidance for use of these foregoing HTPand SFP reaction conditions and the imine reductase polypeptides aregiven in, among others, Tables 3A-3M, and the Examples.

In some embodiments, the polypeptides of the invention can be in theform of fusion polypeptides in which the engineered polypeptides arefused to other polypeptides, such as, by way of example and notlimitation, antibody tags (e.g., myc epitope), purification sequences(e.g., His tags for binding to metals), and cell localization signals(e.g., secretion signals). Thus, the engineered polypeptides describedherein can be used with or without fusions to other polypeptides.

It is to be understood that the polypeptides described herein are notrestricted to the genetically encoded amino acids. In addition to thegenetically encoded amino acids, the polypeptides described herein maybe comprised, either in whole or in part, of naturally-occurring and/orsynthetic non-encoded amino acids. Certain commonly encounterednon-encoded amino acids of which the polypeptides described herein maybe comprised include, but are not limited to: the D-stereomers of thegenetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr);α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovalericacid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine(Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug);N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine(Cha); norleucine (Nle); naphthylalanine (NaI); 2-chlorophenylalanine(Oct); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Oct); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Pat); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

In some embodiments, the engineered polypeptides can be in variousforms, for example, such as an isolated preparation, as a substantiallypurified enzyme, whole cells transformed with gene(s) encoding theenzyme, and/or as cell extracts and/or lysates of such cells. Theenzymes can be lyophilized, spray-dried, precipitated or be in the formof a crude paste, as further discussed below.

In some embodiments, the engineered polypeptides can be provided on asolid support, such as a membrane, resin, solid carrier, or other solidphase material. A solid support can be composed of organic polymers suchas polystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled pore glass (CPG), reverse phase silica or metal, such as goldor platinum. The configuration of a solid support can be in the form ofbeads, spheres, particles, granules, a gel, a membrane or a surface.Surfaces can be planar, substantially planar, or non-planar. Solidsupports can be porous or non-porous, and can have swelling ornon-swelling characteristics. A solid support can be configured in theform of a well, depression, or other container, vessel, feature, orlocation.

In some embodiments, the engineered polypeptides having imine reductaseactivity of the present invention can be immobilized on a solid supportsuch that they retain their improved activity, stereoselectivity, and/orother improved properties relative to reference engineered polypeptidesof SEQ ID NOS:4, 6, and/or 8. In such embodiments, the immobilizedpolypeptides can facilitate the biocatalytic conversion of the ketoneand amine substrate compounds of formula (I) and formula (II) to theamine product compound of formula (III), (e.g., as in conversionreactions (a)-(g) of Table 2), and after the reaction is complete areeasily retained (e.g., by retaining beads on which polypeptide isimmobilized) and then reused or recycled in subsequent reactions. Suchimmobilized enzyme processes allow for further efficiency and costreduction. Accordingly, it is further contemplated that any of themethods of using the imine reductase polypeptides of the presentinvention can be carried out using the same imine reductase polypeptidesbound or immobilized on a solid support.

Methods of enzyme immobilization are well-known in the art. Theengineered polypeptides can be bound non-covalently or covalently.Various methods for conjugation and immobilization of enzymes to solidsupports (e.g., resins, membranes, beads, glass, etc.) are well known inthe art (See e.g., Yi et al., Proc. Biochem., 42: 895-898 [2007]; Martinet al., Appl. Micro. and Biotech., 76: 843-851 [2007]; Koszelewski etal., J. Mol. Cat. B: Enzy., 63: 39-44 [2010]; Truppo et al., Org. Proc.Res. Dev., published online: dx.doi.org/10.1021/op200157c; Hermanson,Bioconjugate Techniques, Second Edition, Academic Press [2008]; Mateo etal., Biotech. Prog., 18:629-34 [2002]; and C. M. Niemeyer [ed.],Bioconjugation Protocols: Strategies and Methods, In Methods inMolecular Biology, Humana Press [2004], each of which are incorporatedby reference herein). Solid supports useful for immobilizing theengineered imine reductases of the present invention include but are notlimited to beads or resins comprising polymethacrylate with epoxidefunctional groups, polymethacrylate with amino epoxide functionalgroups, styrene/DVB copolymer or polymethacrylate with octadecylfunctional groups. Exemplary solid supports useful for immobilizing theengineered imine reductase polypeptides of the present inventioninclude, but are not limited to, chitosan beads, Eupergit C, andSEPABEADs (Mitsubishi), including the following different types ofSEPABEAD: EC-EP, EC-HFA/S, EXA252, EXE119 and EXE120.

In some embodiments, the polypeptides described herein can be providedin the form of kits. The enzymes in the kits may be present individuallyor as a plurality of enzymes. The kits can further include reagents forcarrying out the enzymatic reactions, substrates for assessing theactivity of enzymes, as well as reagents for detecting the products. Thekits can also include reagent dispensers and instructions for use of thekits.

In some embodiments, the kits of the present invention include arrayscomprising a plurality of different imine reductase polypeptides atdifferent addressable position, wherein the different polypeptides aredifferent variants of a reference sequence each having at least onedifferent improved enzyme property. In some embodiments, a plurality ofpolypeptides immobilized on solid supports can be configured on an arrayat various locations, addressable for robotic delivery of reagents, orby detection methods and/or instruments. The array can be used to test avariety of substrate compounds for conversion by the polypeptides. Sucharrays comprising a plurality of engineered polypeptides and methods oftheir use are known in the art (See e.g., WO2009/008908A2).

6.4 Polynucleotides Encoding Engineered Imine Reductases, ExpressionVectors and Host Cells

In another aspect, the present invention provides polynucleotidesencoding the engineered imine reductase polypeptides described herein.The polynucleotides may be operatively linked to one or moreheterologous regulatory sequences that control gene expression to createa recombinant polynucleotide capable of expressing the polypeptide.Expression constructs containing a heterologous polynucleotide encodingthe engineered imine reductase can be introduced into appropriate hostcells to express the corresponding imine reductase polypeptide.

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode the improved imine reductase enzymes. Thus, havingknowledge of a particular amino acid sequence, those skilled in the artcould make any number of different nucleic acids by simply modifying thesequence of one or more codons in a way which does not change the aminoacid sequence of the protein. In this regard, the present inventionspecifically contemplates each and every possible variation ofpolynucleotides that could be made encoding the polypeptides describedherein by selecting combinations based on the possible codon choices,and all such variations are to be considered specifically disclosed forany polypeptide described herein, including the amino acid sequencespresented in Tables 3A-3M and disclosed in the sequence listingincorporated by reference herein as even-numbered sequence identifiersSEQ ID NOS:4-1300.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells. In some embodiments, all codons need not be replaced to optimizethe codon usage of the imine reductases since the natural sequence willcomprise preferred codons and because use of preferred codons may not berequired for all amino acid residues. Consequently, codon optimizedpolynucleotides encoding the imine reductase enzymes may containpreferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90%of codon positions of the full length coding region.

In some embodiments, the polynucleotide comprises a codon optimizednucleotide sequence encoding the naturally occurring imine reductasepolypeptide of SEQ ID NO:2. In some embodiments, the polynucleotide hasa nucleic acid sequence comprising at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identity to the codon optimized nucleicacid sequence of SEQ ID NO:1. The codon optimized sequence of SEQ IDNO:1 enhances expression of the encoded, naturally occurring iminereductase.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference sequence of SEQ IDNO:1, or a complement thereof, and encodes a polypeptide having iminereductase activity.

In some embodiments, as described above, the polynucleotide encodes anengineered polypeptide having imine reductase activity with improvedproperties as compared to SEQ ID NO:2, 4, 6, and/or 8, where thepolypeptide comprises an amino acid sequence having at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identity to a reference sequence selected from even-numberedsequence identifiers SEQ ID NOS:4-1300.

In some embodiments, as described above, the polynucleotide encodes anengineered polypeptide having imine reductase activity with improvedproperties as compared to SEQ ID NO:2, 4, 6, and/or 8, where thepolypeptide comprises an amino acid sequence having at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identity to a reference sequence selected from even-numberedsequence identifiers SEQ ID NOS:4-1300.

In some embodiments, the polynucleotide encodes a imine reductasepolypeptide capable of converting substrate compounds (1a) and (2a) tothe product compound (3a) with improved properties as compared to SEQ IDNO:4, 12 and/or 30, wherein the polypeptide comprises an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to referencesequence SEQ ID NO:4 and one or more residue differences as compared toSEQ ID NO:4 at residue positions selected from: X198, X153, X167, X265,X262, X108, X234, X284, X282, X220, X272, X256, X267, X242, X281, X197,X277, X224, and X143.

In some embodiments, the polynucleotide encodes a imine reductasepolypeptide capable of converting substrate compounds (1b) and (2b) tothe product compound (3b) with improved properties as compared to SEQ IDNO:6, 126 and/or 173, wherein the polypeptide comprises an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to referencesequence SEQ ID NO:6, and at least a combination of residue differencesas compared to SEQ ID NO:6 selected from: X283, X262, X9, X259, X220,X267, X153, X279, X200, X224, X256, X137, X143, X260, X261, X154, X276,and X185.

In some embodiments, the polynucleotide encodes a imine reductasepolypeptide capable of converting substrate compounds (1c) and (2c) tothe product compound (3c) with improved properties as compared to SEQ IDNO:8, wherein the polypeptide comprises an amino acid sequence having atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to reference sequence SEQ IDNO:8, and a combination of residue differences as compared to SEQ IDNO:8, as shown in Table 3G.

In some embodiments, the polynucleotide encodes a imine reductasepolypeptide capable of converting substrate compounds (1d) and (2d) tothe product compound (3d) with improved properties as compared to SEQ IDNO:8, wherein the polypeptide comprises an amino acid sequence having atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to reference sequence SEQ IDNO:8, and a combination of residue differences as compared to SEQ IDNO:8, as shown in Table 3G.

In some embodiments, the polynucleotide encodes a imine reductasepolypeptide capable of converting substrate compounds (1d) and (2c) tothe product compound (3e) with improved properties as compared to SEQ IDNO:8, wherein the polypeptide comprises an amino acid sequence having atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to reference sequence SEQ IDNO:8, and a combination of residue differences as compared to SEQ IDNO:8, as shown in Table 3G.

In some embodiments, the polynucleotide encodes a imine reductasepolypeptide capable of converting substrate compounds (1d) and (2c) tothe product compound (3f) with improved properties as compared to SEQ IDNO:360 and/or 550, wherein the polypeptide comprises an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to referencesequence SEQ ID NO:8, and a combination of residue differences ascompared to SEQ ID NO:8, as shown in Tables 3H and 3I.

In some embodiments, the polynucleotide encodes a imine reductasepolypeptide capable of converting substrate compounds (1d) and (2c) tothe product compound (3g) with improved properties as compared to SEQ IDNO:360 and/or 550, wherein the polypeptide comprises an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to referencesequence SEQ ID NO:8, and at least a combination of residue differencesas compared to SEQ ID NO:8 selected from X12, X31, X37, X44, X46, X48,X55, X56, X65, X82, X93, X98, X108, X137, X138, X141, X142, X143, X153,X154, X155, X156, X159, X166, X168, X173, X177, X178, X184, X185, X195,X197, X199, X200, X201, X203, X205, X206, X210, X213, X215, X216, X218,X220, X221, X223, X226, X234, X239, X242, X245, X251, X253, X256, X257,X259, X260, X261, X262, X263, X265, X266, X267, X268, X269, X273, X274,X277, X278, X279, X280, X281, X282, X283, X284, X285, X289, X290, X291,X292, X293, X294, X330, and X349.

In some embodiments, the polynucleotide encodes an engineered iminereductase polypeptide capable of converting substrate compounds (1) and(2) to the corresponding product compound (3) with improved enzymeproperties as compared to the reference polypeptide of SEQ ID NOS:2, 4,6, 8, 12, 30, 126, 173, 360, and/or 550, wherein the polypeptidecomprises an amino acid sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identityto a reference polypeptide selected from any one of even-numberedsequence identifiers SEQ ID NOS:4-1300, with the proviso that the aminoacid sequence comprises any one of the set of residue differences ascompared to SEQ ID NO:2, contained in any one of the polypeptidesequences of even-numbered sequence identifiers SEQ ID NOS:4-1300, aslisted in Tables 3A-3M.

In some embodiments, the polynucleotide encoding the engineered iminereductase comprises an polynucleotide sequence selected from theodd-numbered sequence identifiers SEQ ID NOS:4-1300.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference polynucleotide sequenceselected from the odd-numbered sequence identifiers SEQ ID NOS:3-1299,or a complement thereof, and encodes a polypeptide having iminereductase activity with one or more of the improved properties describedherein. In some embodiments, the polynucleotide capable of hybridizingunder highly stringent conditions encodes a imine reductase polypeptidethat has an amino acid sequence that comprises one or more residuedifferences as compared to SEQ ID NO:2, 4, 6, and/or 8, at residuepositions selected from: X9, X12, X31, X37, X44, X46, X48, X55, X56,X65, X82, X93, X98, X108, X137, X138, X141, X142, X143, X153, X154,X155, X156, X159, X166, X167, X168, X173, X177, X178, X184, X185, X195,X197, X198, X199, X200, X201, X203, X205, X206, X210, X213, X215, X216,X218, X220, X221, X223, X224, X226, X234, X239, X242, X245, X251, X253,X256, X259, X260, X261, X262, X263, X265, X266, X267, X268, X269, X272,X273, X274, X276, X277, X278, X279, X280, X281, X282, X283, X284, X285,X289, X290, X291, X292, X293, X294, X330, and X349.

In some embodiments, the specific residue differences at these residuepositions are selected from: S284Q/L/H/C, L277A, E272D, S262V,E256V/S/A, I242C, A234C, H220Q, E198D, Y153G/Q/N/R/E, A167T, Q265T,N108C, G282L, P267A, I197M, Q265V, R281N/P/L, E224D, and R143K, relativeto SEQ ID NO:4; or V9G, N137D, R143K, Y153L/M, F154V, A185T, V200S,H220E, E224H, E256C/W, V259I, V259M, K260R, I261R, S262F/N,P267I/N/S/T/Y, G276S, V279A/H/L/N/Y, and V283E/T/W, relative to SEQ IDNO:6; or M12L, L31M, A37P, Q44N, R46S/N, A48G, G55Y/C, L56M, V65L/F,T82V, A93G, Y98W/C, N108S, N137D, M138L, W141V/T/S, C142A, R143W/L,Y153F/E/G/A, F154V/M/L, I155M/S, T156M/Q/G/E/A, Q159L/M/I/E, A1661,K168M, L173M, G177T/A, S178E, Q184L, A185L/G, T195S/R, I197T/L, A199V,V200Y, I201M/L/F/C/A, P203G, P205T/S/N/L/G/D/A, T206V/Q/N/M/L/I/H/D,A210F, C213Y/V/M/L/F, S215E, G216V/S/R, P218Q, H220D, Y221W/M/L/I,T223S, I226Y, C234M/A, A239Q, I242C/V, A245G, N251E, P253D,A256S/L/E/N/K/G, E257S, V259M/L, N260D/K, H261I/E/D, V262S, C263Q/E/T,Q265N, T266P/N, P267V/Q/K, A268N/D, T269H, A273V/T/I, M274V, L277M,P278V, V279F/R/E, L280P/E, R281A/S, G282T, V283Y/w/T, S284P/M/H/I,G285W, L289W/R, N290Y/S/R, T291A, E292V, H293Y/Q/M/L/F, F294A, I330V,and L349P, relative to SEQ ID NO:8.

In some embodiments, the residue differences are selected from E198D,Y153G/Q/N/R, A167T, Q265T/V, N108C, G282L, P267A, I197M, R281N/P/L,E224D, and R143K relative to SEQ ID NO:4; or V9G, N137D, R143K, Y153L/M,A185T, H220E, E224H, E256W/C, V259M/I, K260R, S262N, P267Y/T, G276S,V279A/H/N, V283T/W/E, relative to SEQ ID NO:6; or M12L, L31M, Q44N,R46S/N, A48G, G55Y/C, L56M, V65L/F, T82V, Y98W/C, N137D, W141V/T/S,Y153G/A, I155S, T156Q/G/E/A, Q159M/I/E, A1661, K168M, L173M, G177T/A,Q184L, A185L/G, T195R, I197T/L, A199V, V200Y, I201M/L/F/C/A, P203G,P205T/S/N/L/G/D/A, T206V/Q/N/M/L/I/H/D, A210F, C213Y/V/M/L/F, S215E,G216V/S/R, P218Q, Y221W/M/L/I, I226Y, C234M/A, A239Q, I242V, A245G,N251E, P253D, A256N/K/G, E257S, V259M/L, N260K, H261D, V262S, C263T,Q265N, T266P/N, P267V/Q/K, A268N/D, T269H, A273V/T/I, M274V, L277M,P278V, V279R/E, L280P/E, R281S, G282T, V283Y/W/T, S284I, G285W, L289W/R,N290Y/S/R, T291A, E292V, H293Y/Q/M/L/F, I330V, and L349P, relative toSEQ ID NO:8.

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity at the nucleotide level to areference polynucleotide encoding the engineered imine reductase. Insome embodiments, the reference polynucleotide sequence is selected fromthe odd-numbered sequence identifiers SEQ ID NOS:3-1299.

An isolated polynucleotide encoding an improved imine reductasepolypeptide may be manipulated in a variety of ways to provide forexpression of the polypeptide. In some embodiments, the polynucleotidesencoding the polypeptides can be provided as expression vectors whereone or more control sequences is present to regulate the expression ofthe polynucleotides and/or polypeptides. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art.

In some embodiments, the control sequences include among others,promoters, leader sequence, polyadenylation sequence, propeptidesequence, signal peptide sequence, and transcription terminator.Suitable promoters can be selected based on the host cells used. Forbacterial host cells, suitable promoters for directing transcription ofthe nucleic acid constructs of the present invention, include thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothennophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (Villa-Kamaroff et al., Proc. Natl.Acad. Sci. USA 75: 3727-3731 [1978]), as well as the tac promoter(DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 [1983]). Exemplarypromoters for filamentous fungal host cells, include promoters obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Rhizomucor mieheiaspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusariumoxysporum trypsin-like protease (See e.g., WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof. Exemplary yeast cell promoters can be from the genes can befrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.Other useful promoters for yeast host cells are known in the art (Seee.g., Romanos et al., Yeast 8:423-488 [1992]).

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention. For example, exemplary transcription terminatorsfor filamentous fungal host cells can be obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Aspergillus nigeralpha-glucosidase, and Fusarium oxysporum trypsin-like protease.Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are also known in the art (See e.g., Romanos et al.,supra).

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are known in the art (Seee.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990 [1995]).

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. Any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention. Effectivesignal peptide coding regions for bacterial host cells are the signalpeptide coding regions obtained from the genes for Bacillus NC1B 11837maltogenic amylase, Bacillus stearothennophilus alpha-amylase, Bacilluslicheniformis subtilisin, Bacillus licheniformis beta-lactamase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are known in the art(See e.g., Simonen and Palva, Microbiol. Rev., 57: 109-137 [1993]).Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase. Usefulsignal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are known in theart (See e.g., Romanos et al., supra).

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei asparticproteinase, and Myceliophthora thermophila lactase (See e.g., WO95/33836). Where both signal peptide and propeptide regions are presentat the amino terminus of a polypeptide, the propeptide region ispositioned next to the amino terminus of a polypeptide and the signalpeptide region is positioned next to the amino terminus of thepropeptide region.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide of thepresent invention would be operably linked with the regulatory sequence.

In another aspect, the present invention is also directed to arecombinant expression vector comprising a polynucleotide encoding anengineered imine reductase polypeptide, and one or more expressionregulating regions such as a promoter and a terminator, a replicationorigin, etc., depending on the type of hosts into which they are to beintroduced. The various nucleic acid and control sequences describedabove may be joined together to produce a recombinant expression vectorwhich may include one or more convenient restriction sites to allow forinsertion or substitution of the nucleic acid sequence encoding thepolypeptide at such sites. Alternatively, the nucleic acid sequence ofthe present invention may be expressed by inserting the nucleic acidsequence or a nucleic acid construct comprising the sequence into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector (i.e., avector that exists as an extrachromosomal entity), the replication ofwhich is independent of chromosomal replication (e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificialchromosome). The vector may contain any means for assuringself-replication. Alternatively, the vector may be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. Furthermore, a single vector or plasmid or two or morevectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon may beused.

The expression vector of the present invention preferably contains oneor more selectable markers, which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol (Example 1) or tetracycline resistance.Suitable markers for yeast host cells include, but are not limited toADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for usein a filamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

In another aspect, the present invention provides a host cell comprisinga polynucleotide encoding an improved imine reductase polypeptide of thepresent invention, the polynucleotide being operatively linked to one ormore control sequences for expression of the imine reductase enzyme inthe host cell. Host cells for use in expressing the polypeptides encodedby the expression vectors of the present invention are well known in theart and include but are not limited to, bacterial cells, such as E.coli, Bacillus subtilis, Streptomyces and Salmonella typhimurium cells;fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae orPichia pastoris (ATCC Accession No. 201178)); insect cells such asDrosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS,BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary hostcells are Escherichia coli W3110 (AfhuA) and BL21.

Appropriate culture mediums and growth conditions for theabove-described host cells are well known in the art. Polynucleotidesfor expression of the imine reductase may be introduced into cells byvarious methods known in the art. Techniques include among others,electroporation, biolistic particle bombardment, liposome mediatedtransfection, calcium chloride transfection, and protoplast fusion.

In some embodiments, the polypeptides can be expressed in cell freeexpression systems (See e.g., Kudlicki et al., Cell Free Expression,1^(st) Ed., Landes Biosciences [2007]; and Spirin et al. (eds.), CellFree Protein Synthesis: Methods and Protocols, 1^(st) ed., Wiley-VCH[2007], all of which are incorporated herein by reference).

In the embodiments herein, the improved polypeptides and correspondingpolynucleotides can be obtained using methods used by those skilled inthe art. The engineered imine reductases described herein can beobtained by subjecting the polynucleotide encoding the naturallyoccurring gene encoding the wild-type opine dehydrogenase CENDH (SEQ IDNO:2) or another engineered imine reductase to mutagenesis and/ordirected evolution methods, as discussed above.

For example, mutagenesis and directed evolution methods can be readilyapplied to polynucleotides to generate variant libraries that can beexpressed, screened, and assayed. Mutagenesis and directed evolutionmethods are well known in the art (See e.g., U.S. Pat. Nos. 5,605,793,5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548,6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,277,638,6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883,6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198,6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377,6,365,408, 6,368,861, 6,372,497, 6,376,246, 6,379,964, 6,387,702,6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745,6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468,6,455,253, 6,479,652, 6,482,647, 6,489,146, 6,506,602, 6,506,603,6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467,6,579,678, 6,586,182, 6,602,986, 6,613,514, 6,653,072, 6,716,631,6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297,7,148,054, 7,288,375, 7,421,347, 7,430,477, 7,534,564, 7,620,500,7,620,502, 7,629,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986,7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,873,499,7,904,249, 7,957,912, 8,383,346, 8,504,498, 8,849,575, 8,876,066,8,768,871, and all related non-US counterparts; Ling et al., Anal.Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol., 57:369-74[1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al.,Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7 [1986];Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323[1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999];Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et al.,Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol.,15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A.,94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319[1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad.Sci. USA, 91:10747-10751 [1994]; US Pat. Appln. Publn. Nos.2008/0220990, US 2009/0312196, US2014/0005057, US2014/0214391,US2014/0221216; US2015/0050658, US2015/0133307, US2015/0134315 and allrelated non-US counterparts; WO 95/22625, WO 97/0078, WO 97/35966, WO98/27230, WO 00/42651, WO 01/75767, and WO 2009/152336; all of which areincorporated herein by reference). Other directed evolution proceduresthat find use include, but are not limited to can be used include, amongothers, staggered extension process (StEP), in vitro recombination (Seee.g., Zhao et al., Nat. Biotechnol., 16:258-261 [1998]), mutagenic PCR(See e.g., Caldwell et al., PCR Meth. Appl., 3:S136-S140 [1994]), andcassette mutagenesis (See e.g., Black et al., Proc. Natl. Acad. Sci. USA93:3525-3529 [1996]), all of which are incorporated herein by reference.

The clones obtained following mutagenesis treatment can be screened forengineered imine reductases having one or more desired improved enzymeproperties. For example, where the improved enzyme property desired isincrease activity in the conversion of a ketone of compound (1b) and anamine of compound (2b) to a secondary amine of compound (3b), enzymeactivity may be measured for production of compound (3b). Clonescontaining a polynucleotide encoding a imine reductase with the desiredcharacteristics, e.g., increased production of compound (3b), are thenisolated, sequenced to identify the nucleotide sequence changes (ifany), and used to express the enzyme in a host cell. Measuring enzymeactivity from the expression libraries can be performed using thestandard biochemistry techniques, such as HPLC analysis and/orderivatization of products (pre or post separation), e.g., with dansylchloride or OPA.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides encodingportions of the imine reductase can be prepared by chemical synthesisusing the classical phosphoramidite method (See e.g., Beaucage et al.,Tetra. Lett., 22:1859-69 [1981]), or alternative methods (See e.g.,Matthes et al., EMBO J., 3:801-05 [1984]), as it is typically practicedin automated synthetic methods. According to the phosphoramidite method,oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer,purified, annealed, ligated and cloned in appropriate vectors. Inaddition, essentially any nucleic acid can be obtained from any of avariety of commercial sources. In some embodiments, additionalvariations can be created by synthesizing oligonucleotides containingdeletions, insertions, and/or substitutions, and combining theoligonucleotides in various permutations to create engineered iminereductases with improved properties.

In some embodiments, the present invention provides methods forpreparing engineered imine reductase polypeptides comprising: (a)synthesizing a polynucleotide encoding a polypeptide comprising an aminoacid sequence selected from the even-numbered sequence identifiers SEQID NOS:4-1300, and having one or more residue differences as compared toSEQ ID NO:4, at residue positions selected from X198, X153, X167, X265,X262, X108, X234, X284, X282, X220, X272, X256, X267, X242, X281, X197,X277, X224, and X143; and (b) expressing the imine reductase polypeptideencoded by the polynucleotide. In some embodiments of the method, theresidue differences are selected from E198, Y153, A167, Q265, 5262,N108, A234, 5284, G282, H220, E272, E256, P267, 1242, R281, 1197, L277,E224, and R143, relative to SEQ ID NO:4. In some embodiments of themethod, the residue differences are selected from S284Q/L/H/C, L277A,E272D, S262V, E256V/S/A, I242C, A234C, H220Q, E198D, Y153G/Q/N/R/E,A167T, Q265T, N108C, G282L, P267A, I197M, Q265V, R281N/P/L, E224D, andR143K, relative to SEQ ID NO:4. In some additional embodiments, theresidue differences are selected from X198, X167, and X224, relative toSEQ ID NO:4. In some further embodiments, the residue differences areselected from E198, A167, and E224, relative to SEQ ID NO:4. In someadditional embodiments, the residue differences are selected from E198D,Y153G/Q/N/R, A167T, Q265T/V, N108C, G282L, P267A, I197M, R281N/P/L,E224D, and R143K, relative to SEQ ID NO:4.

In some embodiments, the present invention provides methods forpreparing engineered imine reductase polypeptides comprising: (a)synthesizing a polynucleotide encoding a polypeptide comprising an aminoacid sequence selected from the even-numbered sequence identifiers SEQID NOS:4-1300, and having one or more residue differences as compared toSEQ ID NO:6, at residue positions selected from X283, X262, X9, X259,X220, X267, X153, X279, X200, X224, X256, X137, X143, X260, X261, X154,X276, and X185; and (b) expressing the imine reductase polypeptideencoded by the polynucleotide. In some embodiments of the method, theresidue differences are selected from V283, 5262, V9, V259, H220, P267,Y153, V279, V200, E224, E256, N137, R143, K260, I261, F154, G276, andA185, relative to SEQ ID NO:6. In some embodiments of the method, theresidue differences are selected from V9G, N137D, R143K, Y153L/M, F154V,A185T, V200S, H220E, E224H, E256C/W, V259I, V259M, K260R, I261R,S262F/N, P267I/N/S/T/Y, G276S, V279A/H/L/N/Y, and V283E/T/W, relative toSEQ ID NO:6. In some additional embodiments, the residue differences areselected from X9, and X224, relative to SEQ ID NO:6. In some furtherembodiments, the residue differences are selected from V9 and E224,relative to SEQ ID NO:6. In some additional embodiments, the residuedifferences are selected from V9G, N137D, R143K, Y153L/M, A185T, H220E,E224H, E256W/C, V259M/I, K260R, S262N, P267Y/T, G276S, V279A/H/N, andV283T/W/E, relative to SEQ ID NO:6.

In some embodiments, the present invention provides methods forpreparing engineered imine reductase polypeptides comprising: (a)synthesizing a polynucleotide encoding a polypeptide comprising an aminoacid sequence selected from the even-numbered sequence identifiers SEQID NOS:4-1300, and having one or more residue differences as compared toSEQ ID NO:8, at residue positions selected from X12, X31, X37, X44, X46,X48, X55, X56, X65, X82, X93, X98, X108, X137, X138, X141, X142, X143,X153, X154, X155, X156, X159, X166, X168, X173, X177, X178, X184, X185,X195, X197, X199, X200, X201, X203, X205, X206, X210, X213, X215, X216,X218, X220, X221, X223, X226, X234, X239, X242, X245, X251, X253, X256,X257, X259, X260, X261, X262, X263, X265, X266, X267, X268, X269, X273,X274, X277, X278, X279, X280, X281, X282, X283, X284, X285, X289, X290,X291, X292, X293, X294, X330, and X349; and (b) expressing the iminereductase polypeptide encoded by the polynucleotide. In some embodimentsof the method, the residue differences are selected from M12, L31, A37,Q44, R46, A48, G55, L56, V65, T82, A93, Y98, N108, N137, M138, W141,C142, R143, Y153, F154, I155, T156, Q159, A166, K168, L173, G177, S178,Q184, A185, T195, I197, A199, V200, I201, P203, P205, T206, A210, C213,S215, G216, P218, H220, Y221, T223, I226, C234, A239, I242, A245, N251,P253, A256, E257, V259, N260, H261, V262, C263, Q265, T266, P267, A268,T269, A273, M274, L277, P278, V279, L280, R281, G282, V283, 5284, G285,L289, N290, T291, E292, H293, F294, I330, and L349, relative to SEQ IDNO:8. In some embodiments of the method, the residue differences areselected from M12L, L31M, A37P, Q44N, R46S/N, A48G, G55Y/C, L56M,V65L/F, T82V, A93G, Y98W/C, N108S, N137D, M138L, W141V/T/S, C142A,R143W/L, Y153F/E/G/A, F154V/M/L, I155M/S, T156M/Q/G/E/A, Q159L/M/I/E,A1661, K168M, L173M, G177T/A, S178E, Q184L, A185L/G, T195S/R, I197T/L,A199V, V200Y, I201M/L/F/C/A, P203G, P205T/S/N/L/G/D/A,T206V/Q/N/M/L/I/H/D, A210F, C213Y/V/M/L/F, S215E, G216V/S/R, P218Q,H220D, Y221W/M/L/I, T223S, I226Y, C234M/A, A239Q, I242C/V, A245G, N251E,P253D, A256S/L/E/N/K/G, E257S, V259M/L, N260D/K, H261I/E/D, V262S,C263Q/E/T, Q265N, T266P/N, P267V/Q/K, A268N/D, T269H, A273V/T/I, M274V,L277M, P278V, V279F/R/E, L280P/E, R281A/S, G282T, V283Y/W/T,S284P/M/H/I, G285W, L289W/R, N290Y/S/R, T291A, E292V, H293Y/Q/M/L/F,F294A, I330V, and L349P, relative to SEQ ID NO:8. In some additionalembodiments, the residue differences are selected from X31, X44, X46,X48, X55, X56, X98, X166, X168, X173, X199, X203, X205, X206, X210,X213, X215, X216, X218, X239, X245, X251, X268, X269, X280, X285, X289,X290, X293, X330, and X349, relative to SEQ ID NO:8. In some furtherembodiments, the residue differences are selected from L31, Q44, R46,A48, G55, L56, Y98, A166, K168, L173, A199, P203, P205, T206, A210,C213, S215, G216, P218, A239, A245, N251, A268, T269, L280, G285, L289,N290, H293, I330, and L349, relative to SEQ ID NO:8. In some additionalembodiments, the residue differences are selected from M12L, L31M, Q44N,R46S/N, A48G, G55Y/C, L56M, V65L/F, T82V, Y98W/C, N137D, W141V/T/S,Y153G/A, I155S, T156Q/G/E/A, Q159M/I/E, A1661, K168M, L173M, G177T/A,Q184L, A185L/G, T195R, I197T/L, A199V, V200Y, I201M/L/F/C/A, P203G,P205T/S/N/L/G/D/A, T206V/Q/N/M/L/I/H/D, A210F, C213Y/V/M/L/F, S215E,G216V/S/R, P218Q, Y221W/M/L/I, I226Y, C234M/A, A239Q, I242V, A245G,N251E, P253D, A256N/K/G, E257S, V259M/L, N260K, H261D, V262S, C263T,Q265N, T266P/N, P267V/Q/K, A268N/D, T269H, A273V/T/I, M274V, L277M,P278V, V279R/E, L280P/E, R281S, G282T, V283Y/W/T, S284I, G285W, L289W/R,N290Y/S/R, T291A, E292V, H293Y/Q/M/L/F, I330V, and L349P, relative toSEQ ID NO:8.

In some embodiments of the method, the polynucleotide encodes anengineered imine reductase that has optionally one or several (e.g., upto 3, 4, 5, or up to 10) amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the number of amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,30, 35, 40, 45, or 50 amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the substitutions can beconservative or non-conservative substitutions.

In another aspect, the present invention provides methods ofmanufacturing the engineered imine reductase polypeptides, where themethod can comprise culturing a host cell capable of expressing apolynucleotide encoding the imine reductase polypeptide under conditionssuitable for expression of the polypeptide. The method can furthercomprise isolating or purifying the expressed imine reductasepolypeptide, as described herein.

In some embodiments, the method for preparing or manufacturing theengineered imine reductase polypeptides further comprises the step ofisolating the polypeptide. The engineered polypeptides can be expressedin appropriate cells, as described above, and isolated (or recovered)from the host cells, the culture medium, and/or expression medium usingany one or more of the well known techniques used for proteinpurification, including, among others, lysozyme treatment, sonication,filtration, salting-out, ultra-centrifugation, and chromatography.Suitable solutions for lysing and the high efficiency extraction ofproteins from bacteria, such as E. coli, are commercially available,such as CelLytic B™ (Sigma-Aldrich, St. Louis M)O. Chromatographictechniques for isolation of the imine reductase polypeptides include,among others, reverse phase chromatography high performance liquidchromatography, ion exchange chromatography, gel electrophoresis, andaffinity chromatography.

In some embodiments, the non-naturally occurring polypeptides of theinvention can be prepared and used in various forms including but notlimited to crude extracts (e.g., cell-free lysates), powders (e.g.,shake-flask powders), lyophilizates, and substantially pure preparations(e.g., DSP powders), as further illustrated in the Examples below.

In some embodiments, the engineered polypeptides can be prepared andused in purified form, for example a substantially purified form.Generally, conditions for purifying a particular polypeptide willdepend, in part, on factors such as net charge, hydrophobicity,hydrophilicity, molecular weight, molecular shape, etc., and will beapparent to those having skill in the art. To facilitate purification,it is contemplated that in some embodiments the engineered polypeptidescan be expressed as fusion proteins with purification tags, such asHis-tags having affinity for metals, or antibody tags for binding toantibodies, e.g., myc epitope tag.

In some embodiments, affinity techniques may be used to isolate theimproved imine reductase enzymes. For affinity chromatographypurification, any antibody which specifically binds the imine reductasepolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with a imine reductase polypeptide, or a fragmentthereof. The imine reductase polypeptide or fragment may be attached toa suitable carrier, such as BSA, by means of a side chain functionalgroup or linkers attached to a side chain functional group. In someembodiments, the affinity purification can use a specific ligand boundby the imine reductase, such as poly(L-proline) or dye affinity column(see, e.g., EP0641862; and Stellwagen, in Current Protocols in ProteinScience, Unit 9.2-9.2.16 [2001]).

6.5 Methods of Using the Engineered Imine Reductase Enzymes

In another aspect, the engineered polypeptides having imine reductaseactivity described herein can be used in a process for converting acompound of formula (I) and a compound of formula (II) to a secondary ortertiary amine compound of formula (III) as described above andillustrated in Scheme 1. Generally, such a biocatalytic process forcarrying out the reductive amination reaction of Scheme 1 comprisescontacting or incubating the ketone and amine substrate compounds withan engineered polypeptide having imine reductase activity of the presentinvention in the presence of a cofactor, such as NADH or NADPH, underreaction conditions suitable for formation of the amine product compoundof formula (III).

In some embodiments, the imine reductases can be used in a process forpreparing a secondary or tertiary amine product compound of formula(III),

wherein, R¹ and R² groups are independently selected from optionallysubstituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl; and optionally R¹ andR² are linked to form a 3-membered to 10-membered ring; R³ and R⁴ groupsare independently selected from a hydrogen atom, and optionallysubstituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl, with the proviso thatboth R³ and R⁴ cannot be hydrogen; and optionally R³ and R⁴ are linkedto form a 3-membered to 10-membered ring; and optionally, the carbonatom and/or the nitrogen indicated by * is chiral. The process comprisescontacting a ketone compound of formula (I),

wherein R¹, and R² are as defined above; and an amine compound offormula (II),

wherein R³, and R⁴ are as defined above; with an engineered polypeptidehaving imine reductase activity in presence of a cofactor under suitablereaction conditions.

As illustrated by the reactions in Table 2, and Tables 3A-3M, theengineered polypeptides having imine reductase activity of the presentinvention have activity with, or can be further engineered to haveactivity with, a wide range of amine substrate compounds of formula (II)in a process for preparing compound of formula (III). Accordingly, insome embodiments of the above biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), thecompound of formula (II) can be a primary amine wherein at least one ofR³ and R⁴ is hydrogen, whereby the product of formula (III) is asecondary amine compound. In some embodiments of the process, neither R³or R⁴ is hydrogen and the compound of formula (II) is a secondary amine,whereby the compound of formula (III) is tertiary amine. In someembodiments of the process, the compound of formula (II) is a secondaryamine and R³ or R⁴ are different, whereby the nitrogen atom indicatedby * of the amine compound of formula (III) is chiral. Further, in someembodiments, one stereoisomer of the chiral amine compound of formula(III) is formed stereoselectively, and optionally formed highlystereoselectively (e.g., in at least about 85% stereomeric excess).

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R³and R⁴ groups of the compound of formula (II) are linked to form a3-membered to 10-membered ring. In some embodiments, the ring is a5-membered to 8-membered is an optionally substituted cycloalkyl, aryl,arylalkyl, heterocycloalkyl, heteroaryl, or heteroarylalkyl ring.

In some embodiments of the biocatalytic process for preparing an amineproduct compound of formula (III), the compound of formula (II) is aprimary amine, wherein R³ group is hydrogen, and R⁴ is selected fromoptionally substituted (C₁-C₆)alkyl, (C₁-C₆)alkenyl, (C₁-C₆)alkynyl,(C₁-C₆)carboxyalkyl, (C₁-C₆)aminoalkyl, (C₁-C₆)haloalkyl, and(C₁-C₆)alkylthioalkyl. In some embodiments, the R⁴ group is selectedfrom optionally substituted (C₁-C₆)alkyl, (C₁-C₆)carboxyalkyl, and(C₁-C₆)aminoalkyl. In some embodiments, the R⁴ group is optionallysubstituted (C₁-C₆)alkyl, or (C₁-C₆)carboxyalkyl. In some embodiments,the compound of formula (II) is selected from methylamine,dimethylamine, isopropylamine, butylamine, and isobutylamine. In someembodiments, the amine substrate compound R³ group is hydrogen, and R⁴is selected from optionally substituted (C₄-C₈)cycloalkyl,(C₄-C₈)heterocycloalkyl, (C₄-C₈)aryl, (C₄-C₈)arylalkyl,(C₄-C₈)heteroaryl, and (C₄-C₈)heteroarylalkyl. In some embodiments, theamine substrate compound R³ group is hydrogen, and R⁴ is selected fromoptionally substituted (C₄-C₈)aryl, (C₄-C₈)arylalkyl, (C₄-C₈)heteroaryl,and (C₄-C₈)heteroarylalkyl. In some embodiments, the amine substratecompound R³ group is hydrogen, and R⁴ is optionally substituted(C₄-C₈)aryl. In some embodiments, the compound of formula (II) isoptionally substituted aniline.

As illustrated by the reactions in Table 2, and Tables 3A-3M theengineered polypeptides having imine reductase activity of the presentinvention have activity with, or can be further engineered to haveactivity with, a wide range of ketone substrate compounds of formula (I)in a process for preparing compound of formula (III). In someembodiments, the R¹ and R² groups of the ketone substrate of compound(I) are different, whereby the carbon atom indicated by * of the aminecompound of formula (III) is chiral. Further, in some embodiments of theprocess, one stereoisomer of the chiral amine compound of formula (III)is formed stereoselectively, and optionally formed highlystereoselectively (e.g., in at least about 85% stereomeric excess).

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹and R² groups of the compound of formula (I) are linked to form a3-membered to 10-membered ring. In some embodiments, the ring is anoptionally substituted cycloalkyl, aryl, arylalkyl, heterocycloalkyl,heteroaryl, or heteroarylalkyl ring. In some embodiments of the process,the compound of formula (I) is selected from optionally substitutedcyclobutanone, cyclopentanone, cyclohexanone, and cycloheptanone.

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹and R² groups of the compound of formula (I) are independently selectedfrom optionally substituted (C₁-C₆)alkyl, (C₁-C₆)alkenyl,(C₁-C₆)alkynyl, (C₁-C₆)carboxyalkyl, (C₁-C₆)aminoalkyl,(C₁-C₆)haloalkyl, and (C₁-C₆)alkylthioalkyl.

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹group of the compound of formula (I) is selected from optionallysubstituted (C₁-C₆)alkyl, (C₁-C₆)alkenyl, (C₁-C₆)alkynyl,(C₁-C₆)carboxyalkyl, (C₁-C₆)aminoalkyl, (C₁-C₆)haloalkyl, and(C₁-C₆)alkylthioalkyl; and the R² group of the compound of formula (I)is selected from optionally substituted (C₄-C₈)cycloalkyl,(C₄-C₈)heterocycloalkyl, (C₄-C₈)arylalkyl, (C₄-C₈)heteroaryl, and(C₄-C₈)heteroarylalkyl.

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R′group of the compound of formula (I) is carboxy. In some embodiments,the compound of formula (I) is a 2-keto-acid selected from pyruvic acid,2-oxo-propanoic acid, 2-oxo-butanoic acid, 2-oxo-pentanoic acid,2-oxo-hexanoic acid.

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹group of the compound of formula (I) is a hydrogen atom, and thecompound of formula (I) is an aldehyde. In such embodiments, the R¹group of the compound of formula (I) is selected from optionallysubstituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl.

As illustrated by the compounds of formulas (I), (II), and (III), listedfor the reactions in Table 2, in some embodiments of the abovebiocatalytic process for preparing a secondary or tertiary amine productcompound of formula (III), the product compound of formula (III)comprises a compound selected from group consisting of: compound (3a),compound (3b), compound (3c), compound (3d), compound (3e), compound(3f), and compound (3g). In some embodiments of the process, thecompound of formula (I) comprises a compound selected from groupconsisting of: compound (1a), compound (1b), compound (1c), and compound(1d). In some embodiments of the process, the compound of formula (II)comprises a compound selected from group consisting of: compound (2a),compound (2b), and compound (2c).

It is also contemplated that in some embodiments the process forpreparing an amine product compound of formula (III) catalyzed by anengineered polypeptide having imine reductase activity of the presentinvention comprises an intramolecular reaction, wherein the compound offormula (I) and the compound of formula (II) are groups on the same,single molecule. Thus, in some embodiments, at least one of R¹ and R² ofthe ketone compound of formula (I) is linked to at least one of R³ andR⁴ of the amine compound of formula (II), and the method comprisescontacting the single compound with a ketone group of formula (I) linkedto an amine group of formula (II) with an engineered polypeptide of thepresent invention under suitable reaction conditions. Illustrativeintramolecular reactions include but are not limited to reactions ofSchemes 2-5 shown below in Table 4, wherein groups R₁ and R₃ are asdefined above for groups R¹ and R³, and group R₅ is selected from ahydrogen atom, and optionally substituted alkyl, alkenyl, alkynyl,alkoxy, carboxy, aminocarbonyl, heteroalkyl, heteroalkenyl,heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl,cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, andheteroarylalkyl.

TABLE 4 Scheme 2

Scheme 3

Scheme 4

Scheme 5

Without being bound by theory, it is believed that in most cases thebiocatalytic reaction of Scheme 1 involves the formation of anintermediate imine compound (e.g., an iminium intermediate) which isthen further reduced by the enzyme to the final secondary or tertiaryamine product compound of formula (III). It is also contemplated that insome embodiments, the process for preparing an amine product compound offormula (III) catalyzed by an engineered polypeptide having iminereductase activity of the present invention comprises contacting anengineered imine reductase polypeptide of the present invention with aketone compound of formula (I) and a primary amine compound of formula(II), whereby an imine intermediate is formed which then undergoes anintramolecular asymmetric cyclization reaction to yield a cyclicsecondary or tertiary hydroxyamine intermediate which undergoes hydroxylelimination to give a second imine (or enamine) intermediate. Thissecond imine (or enamine) is subsequently is then reduced in situ by theengineered imine reductase polypeptide of the present invention to yieldthe final cyclic amine product. Illustrative reactions involvingasymmetric cyclization through a hydroxyamine intermediate include butare not limited to reactions of Schemes 6-9 shown below in Table 5,wherein groups R₁ and R₃ are as defined above for groups R¹ and R³, andgroups R₅, R₆, and R₇ are independently selected from a hydrogen atom,and optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy,aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl,aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl.

TABLE 5 Scheme 6

Scheme 7

Scheme 8

Scheme 9

Without being bound by theory, it is believed that the engineeredpolypeptides having imine reductase activity (IREDs) mediate not onlythe formation of the imine and/or hydroxyamine intermiates as shown inthe reactions of Schemes 2-9 but also the conversion of the imineintermediates to the final amine product compound of formula (III)depicted by the second reaction arrow.

Generally, imine compounds are less stable than amine compounds andsusceptible to undesirable oxidation reactions. It is contemplated,however, that in some embodiments of the processes of the presentinvention, an imine compound, or an enamine compound which cantautomerize to form an imine, can form from a ketone of formula (I) andan amine compound of formula (II) absent the presence of an enzyme, andthen be contacted with an engineered polypeptide of the presentinvention to catalyze its conversion of the final to a secondary ortertiary amine product compound of formula (III). For example, an imineor enamine intermediate compound first can be formed by combining aketone of formula (I) and an amine compound of formula (II) as shown inSchemes 6-9 but without the presence of an IRED (i.e., an engineeredpolypeptide having imine reductase activity). The imine compound formeddirectly or from tautomerization of an enamine compound can then becontacted with an engineered polypeptide having imine reductase activityto catalyze the conversion to the final amine product compound offormula (III). In some embodiments, it is contemplated that the imine orenamine intermediate compound, where suitably stable, can be isolatedbefore carrying out a step of contacting it with an engineeredpolypeptide having imine reductase activity. Thus, it is contemplatedthat in some embodiments of the process, an imine or enamine compound isformed first from the compounds of formula (I) and formula (II), orthrough the intramolecular reaction of compound having a ketone grouplinked to an amine group, and then this imine or enamine compound iscontacted with an engineered polypeptide having imine reductase activityto form an amine product compound of formula (III).

In some embodiments, a stable imine or enamine compound may be obtained(i.e., without first reacting a ketone compound of formula (I) and anamine compound of formula (II)) and used directly as a substrate with anIRED. It is contemplated that in such embodiments, the biocatalyticprocess is carried out wherein there is only a single substrate which isa stable imine or enamine compound, and this compound is contacted withan engineered polypeptide having imine reductase activity of the presentinvention which catalyzes the reduction of the stable imine compound toform a secondary compound of formula (III). In such a reaction, thestereoselectivity of the engineered polypeptide can mediate theformation of a chiral center adjacent to the amine group of the compoundof formula (III). Table 6 (below) lists three examples of stable iminecompounds that can undergo chiral reduction in a biocatalytic processwith an engineered polypeptide of the present invention to produceintermediate compounds for the synthesis of the pharmaceuticalssolifenacin and tadalafil, and for the synthesis of the pharmaceuticalcompound, dexmethylphenidate.

TABLE 6 Stable Imine Compounds That Can Undergo Chiral Reduction in aBiocatalytic Process Imine or Enamine Product Compound SubstrateCompound of formula (III)

Alternatively, it is also contemplated that any of the product compoundsof formula (III) produced via an IRED catalyzed reaction of an isolatedimine or enamine substrate compound (as shown in Table 6) could also beproduced via an IRED-catalyzed intramolecular reaction (like thoseillustrated in Table 4) using as substrate the open-chain version of theimine or enamine substrate compounds. Accordingly, each of the productcompounds of Table 6 could also be made using the intermolecularsubstrate shown in Table 7 with an engineered polypeptide having iminereductase activity of the present invention.

TABLE 7 Alternative Intermolecular Substrate Compounds and ProductCompounds Intermolecular Substrate Compound Product Compound (comprisingformula (I) and formula (II)) of formula (III)

There are numerous active pharmaceutical ingredient compounds thatinclude a secondary or tertiary amine group which could be produced viaa biocatalytic reductive amination using an engineered polypeptidehaving imine reductase activity of the present invention, and/or anengineered polypeptide produced by further directed evolution of anengineered polypeptide of the present invention. For example, Table 8lists various product compounds of formula (III) that are known activepharmaceutical ingredient compounds, or intermediate compounds usefulfor the synthesis of active pharmaceutical ingredient compounds, thatcould be produced using an engineered polypeptide having imine reductaseactivity of the present invention with the corresponding substratescompounds of formula (I) and/or formula (II).

TABLE 8 Substrate and Product Compounds Substrate Compound SubstrateCompound Product Compound of formula (I) of formula (II) of formula(III)

Me₂NH

Me₂NH

Me₂NH

In some embodiments of the above biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), theengineered polypeptide having imine reductase activity is derived from anaturally occurring opine dehydrogenase. In some embodiments, theengineered polypeptide having imine reductase activity is an engineeredpolypeptide derived from the opine dehydrogenase from Arthrobacter sp.strain 1C of SEQ ID NO:2, as disclosed herein, and exemplified by theengineered imine reductase polypeptides of even numbered sequenceidentifiers SEQ ID NOS:4-1300.

Any of the engineered imine reductases described herein can be used inthe above biocatalytic processes for preparing a secondary or tertiaryamine compound of formula (III). By way of example and withoutlimitation, in some embodiments, the process can use an engineered iminereductase polypeptide comprising an amino acid sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identity to a reference sequence selected fromeven-numbered sequence identifiers SEQ ID NOS:4-1300, comprising animine reductase polypeptide that has an amino acid sequence thatcomprises one or more (i.e., at least one, at least two, at least three,etc.) residue differences as compared to SEQ ID NO:2, 4, 6, and/or 8, atresidue positions selected from: X9, X12, X31, X37, X44, X46, X48, X55,X56, X65, X82, X93, X98, X108, X137, X138, X141, X142, X143, X153, X154,X155, X156, X159, X166, X167, X168, X173, X177, X178, X184, X185, X195,X197, X198, X199, X200, X201, X203, X205, X206, X210, X213, X215, X216,X218, X220, X221, X223, X224, X226, X234, X239, X242, X245, X251, X253,X256, X259, X260, X261, X262, X263, X265, X266, X267, X268, X269, X272,X273, X274, X276, X277, X278, X279, X280, X281, X282, X283, X284, X285,X289, X290, X291, X292, X293, X294, X330, and X349.

In some embodiments, the specific residue differences at these residuepositions are selected from: S284Q/L/H/C, L277A, E272D, S262V,E256V/S/A, I242C, A234C, H220Q, E198D, Y153G/Q/N/R/E, A167T, Q265T,N108C, G282L, P267A, I197M, Q265V, R281N/P/L, E224D, and R143K, relativeto SEQ ID NO:4; or V9G, N137D, R143K, Y153L/M, F154V, A185T, V200S,H220E, E224H, E256C/W, V259I, V259M, K260R, I261R, S262F/N,P267I/N/S/T/Y, G276S, V279A/H/L/N/Y, and V283E/T/W, relative to SEQ IDNO:6; or M12L, L31M, A37P, Q44N, R46S/N, A48G, G55Y/C, L56M, V65L/F,T82V, A93G, Y98W/C, N108S, N137D, M138L, W141V/T/S, C142A, R143W/L,Y153F/E/G/A, F154V/M/L, I155M/S, T156M/Q/G/E/A, Q159L/M/I/E, A1661,K168M, L173M, G177T/A, S178E, Q184L, A185L/G, T195S/R, I197T/L, A199V,V200Y, I201M/L/F/C/A, P203G, P205T/S/N/L/G/D/A, T206V/Q/N/M/L/I/H/D,A210F, C213Y/V/M/L/F, S215E, G216V/S/R, P218Q, H220D, Y221W/M/L/I,T223S, I226Y, C234M/A, A239Q, I242C/V, A245G, N251E, P253D,A256S/L/E/N/K/G, E257S, V259M/L, N260D/K, H261I/E/D, V262S, C263Q/E/T,Q265N, T266P/N, P267V/Q/K, A268N/D, T269H, A273V/T/I, M274V, L277M,P278V, V279F/R/E, L280P/E, R281A/S, G282T, V283Y/W/T, S284P/M/H/I,G285W, L289W/R, N290Y/S/R, T291A, E292V, H293Y/Q/M/L/F, F294A, 1330V,and L349P, relative to SEQ ID NO:8.

In some embodiments, the residue differences are selected from E198D,Y153G/Q/N/R, A167T, Q265T/V, N108C, G282L, P267A, I197M, R281N/P/L,E224D, and R143K relative to SEQ ID NO:4; or V9G, N137D, R143K, Y153L/M,A185T, H220E, E224H, E256W/C, V259M/I, K260R, S262N, P267Y/T, G276S,V279A/H/N, V283T/W/E, relative to SEQ ID NO:6; or M12L, L31M, Q44N,R46S/N, A48G, G55Y/C, L56M, V65L/F, T82V, Y98W/C, N137D, W141V/T/S,Y153G/A, I155S, T156Q/G/E/A, Q159M/I/E, A1661, K168M, L173M, G177T/A,Q184L, A185L/G, T195R, I197T/L, A199V, V200Y, I201M/L/F/C/A, P203G,P205T/S/N/L/G/D/A, T206V/Q/N/M/L/I/H/D, A210F, C213Y/V/M/L/F, S215E,G216V/S/R, P218Q, Y221W/M/L/I, I226Y, C234M/A, A239Q, I242V, A245G,N251E, P253D, A256N/K/G, E257S, V259M/L, N260K, H261D, V262S, C263T,Q265N, T266P/N, P267V/Q/K, A268N/D, T269H, A273V/T/I, M274V, L277M,P278V, V279R/E, L280P/E, R281S, G282T, V283Y/W/T, S284I, G285W, L289W/R,N290Y/S/R, T291A, E292V, H293Y/Q/M/L/F, 1330V, and L349P, relative toSEQ ID NO:8.

In some embodiments of the above processes, the exemplary iminereductases capable of carrying out the conversion reactions (a)-(g) ofTable 2 disclosed herein, find use. This includes the engineeredpolypeptides disclosed herein comprising an amino acid sequence selectedfrom even-numbered sequence identifiers SEQ ID NOS:4-1300. Guidance onthe choice and use of the engineered imine reductases is provided in thedescriptions herein, for example Tables 3A-3M, and the Examples.

In the embodiments herein and illustrated in the Examples, variousranges of suitable reaction conditions that can be used in theprocesses, include but are not limited to, substrate loading, cofactorloading, polypeptide loading, pH, temperature, buffer, solvent system,reaction time, and/or conditions with the polypeptide immobilized on asolid support. Further suitable reaction conditions for carrying out theprocess for biocatalytic conversion of substrate compounds to productcompounds using an engineered imine reductase polypeptide describedherein can be readily optimized in view of the guidance provided hereinby routine experimentation that includes, but is not limited to,contacting the engineered imine reductase polypeptide and ketone andamine substrate compounds of interest under experimental reactionconditions of concentration, pH, temperature, and solvent conditions,and detecting the product compound.

Generally, in the processes of the present invention, suitable reactionconditions include the presence of cofactor molecule which can act as anelectron donor in the reduction reaction carried out by the iminereductase. In some embodiments, the cofactor is selected from (but notlimited to) NADP (nicotinamide adenine dinucleotide phosphate), NADPH(the reduced form of NADP⁺), NAD (nicotinamide adenine dinucleotide) andNADH (the reduced form of NAD⁺). Generally, the reduced form of thecofactor is added to the enzyme reaction mixture. Accordingly, in someembodiments, the processes are carried out in presence of a cofactorselected from NADPH and NADH (these two cofactors are also referred toherein collectively as “NAD(P)H”). In some embodiments, the electrondonor is NADPH cofactor. In some embodiments, the process can be carriedout wherein the reaction conditions comprise an NADH or NADPH cofactorconcentration of about 0.03 to about 1 g/L, 0.03 to about 0.8 g/L, about0.03 to about 0.5 g/L, about 0.05 to about 0.3 g/L, about 0.05 to about0.2 g/L, or about 0.1 to about 0.2 g/L. In some embodiments, the processis carried out under NADH or NADPH cofactor concentration of about 1g/L, about 0.8 g/L, about 0.5 g/L, about 0.3 g/L, about 0.2 g/L, about0.1 g/L, about 0.05 g/L, or about 0.03 g/L.

In some embodiments of the process, an optional cofactor recyclingsystem, also referred to as a cofactor regeneration system, can be usedto regenerate cofactor NADPH/NADH from NADP+/NAD+ produced in theenzymatic reaction. A cofactor regeneration system refers to a set ofreactants that participate in a reaction that reduces the oxidized formof the cofactor (e.g., NADP to NADPH). Cofactors oxidized by thepolypeptide reduction of the keto substrate are regenerated in reducedform by the cofactor regeneration system. Cofactor regeneration systemscomprise a stoichiometric reductant that is a source of reducinghydrogen equivalents and is capable of reducing the oxidized form of thecofactor. The cofactor regeneration system may further comprise acatalyst, for example an enzyme catalyst that catalyzes the reduction ofthe oxidized form of the cofactor by the reductant. Cofactorregeneration systems to regenerate NADH or NADPH from NAD⁺ or NADP⁺,respectively, are known in the art and can be used in the methodsdescribed herein.

Suitable exemplary cofactor regeneration systems that may be employed inthe imine reductase processes of the present invention include, but arenot limited to, formate and formate dehydrogenase, glucose and glucosedehydrogenase, glucose-6-phosphate and glucose-6-phosphatedehydrogenase, a secondary alcohol and alcohol dehydrogenase, phosphiteand phosphite dehydrogenase, molecular hydrogen and hydrogenase, and thelike. These systems may be used in combination with either NADP⁺/NADPHor NAD⁺/NADH as the cofactor. Electrochemical regeneration usinghydrogenase may also be used as a cofactor regeneration system (Seee.g., U.S. Pat. Nos. 5,538,867 and 6,495,023, both of which areincorporated herein by reference). Chemical cofactor regenerationsystems comprising a metal catalyst and a reducing agent (for example,molecular hydrogen or formate) may also be suitable (See e.g., WO2000/053731, which is incorporated herein by reference).

In some embodiments, the co-factor regenerating system comprises aformate dehydrogenase, which is a NAD⁺ or NADP⁺-dependent enzyme thatcatalyzes the conversion of formate and NAD or NADP⁺ to carbon dioxideand NADH or NADPH, respectively. Formate dehydrogenases suitable for useas cofactor regenerating systems in the imine reductase processesdescribed herein include naturally occurring and non-naturally occurringformate dehydrogenases. Suitable formate dehydrogenases include, but arenot limited to those currently known in the art (See e.g., WO2005/018579, incorporated herein by reference). In some embodiments, theformate dehydrogenase used in the process is FDH-101, which commerciallyavailable (Codexis, Inc., Redwood City, Calif., USA). Formate may beprovided in the form of a salt, typically an alkali or ammonium salt(for example, HCO₂Na, KHCO₂NH₄, and the like), in the form of formicacid, typically aqueous formic acid, or mixtures thereof. A base orbuffer may be used to provide the desired pH.

In some embodiments, the cofactor recycling system comprises glucosedehydrogenase (GDH), which is a NAD⁺ or NADP⁺-dependent enzyme thatcatalyzes the conversion of D-glucose and NAD⁺ or NADP⁺ to gluconic acidand NADH or NADPH, respectively. Glucose dehydrogenases suitable for usein the practice of the imine reductase processes described hereininclude naturally occurring glucose dehydrogenases as well asnon-naturally occurring glucose dehydrogenases. Naturally occurringglucose dehydrogenase encoding genes have been reported in theliterature (e.g., the Bacillus subtilis 61297 GDH gene, B. cereus ATCC14579 and B. megaterium). Non-naturally occurring glucose dehydrogenasesare generated using any suitable method known in the art (e.g.,mutagenesis, directed evolution, and the like; See e.g., WO 2005/018579,and US Pat. Appln. Publ. Nos. 2005/0095619 and 2005/0153417, each ofwhich is incorporated by reference). In some embodiments, the glucosedehydrogenase used in the process is CDX-901 or GDH-105, each of whichcommercially available (Codexis, Inc., Redwood City, Calif., USA).

In some embodiments, the co-factor regenerating system comprises analcohol dehydrogenase or ketoreductase, which is an NAD⁺ orNADP⁺-dependent enzyme that catalyzes the conversion of a secondaryalcohol and NAD⁺ or NADP⁺ to a ketone and NADH or NADPH, respectively.Suitable secondary alcohols useful in cofactor regenerating systemsinclude lower secondary alkanols and aryl-alkyl carbinols, including butnot limited to, isopropanol, 2-butanol, 3-methyl-2-butanol, 2-pentanol,3-pentanol, 3,3-dimethyl-2-butanol, and the like. Alcohol dehydrogenasessuitable for use as cofactor regenerating systems in the processesdescribed herein include naturally occurring and non-naturally occurringketoreductases. Naturally occurring alcohol dehydrogenase/ketoreductaseinclude known enzymes from, by way of example and not limitation,Thermoanerobium brockii, Rhodococcus erythropolis, Lactobacillus kefir,and Lactobacillus brevis, and non-naturally occurring alcoholdehydrogenases include engineered alcohol dehydrogenases derivedtherefrom. In some embodiments, non-naturally occurring ketoreductasesengineered for thermo- and solvent stability can be used. Suchketoreductases include those described herein, as well as others knownin the art (See e.g., US Pat. Appln. Publ. Nos. 20080318295A1, US20090093031A1, US 20090155863A1, US 20090162909A1, US 20090191605A1, US20100055751A1, WO/2010/025238A2, WO/2010/025287A2 and US 20100062499A1;each of which is incorporated by reference herein).

The concentration of the ketone and amine substrate compounds in thereaction mixtures can be varied, taking into consideration, for example,the desired amount of product compound, the effect of substrateconcentration on enzyme activity, stability of enzyme under reactionconditions, and the percent conversion of the substrates to the product.In some embodiments, the suitable reaction conditions comprise asubstrate compound loading of at least about 0.5 to about 200 g/L, 1 toabout 200 g/L, 5 to about 150 g/L, about 10 to about 100 g/L, 20 toabout 100 g/L or about 50 to about 100 g/L. In some embodiments, thesuitable reaction conditions comprise loading of each of the ketone andamine substrate compounds of at least about 0.5 g/L, at least about 1g/L, at least about 5 g/L, at least about 10 g/L, at least about 15 g/L,at least about 20 g/L, at least about 30 g/L, at least about 50 g/L, atleast about 75 g/L, at least about 100 g/L, at least about 150 g/L or atleast about 200 g/L, or even greater. The values for substrate loadingsprovided herein are based on the molecular weight of compound (1d),however it also contemplated that the equivalent molar amounts of otherketone and amine substrates, such as ketone substrate compounds(1a)-(1d), and amine substrate compounds (2a)-(2d), could be used, aswell as equimolar amounts of hydrates or salts of any of these compoundscan be used in the process. It is also contemplated that in someembodiments, the suitable reaction conditions comprise loading of eachof the ketone and amine substrate compounds in terms of molarconcentrations equivalent to the above g/L concentrations for compound(1d). Thus, reaction conditions can comprise a substrate loading of eachof the ketone and amine substrate compounds of at least about 5 mM, atleast about 10 mM, at least about 25 mM, at least about 50 mM, at leastabout 75 mM, at least about 100 mM, or even greater. In addition, it iscontemplated that substrate compounds covered by formulas (I) and (II),can be used in the same ranges of amounts as those used for compound(1d).

In carrying out the imine reductase mediated processes described herein,the engineered polypeptide may be added to the reaction mixture in theform of a purified enzyme, partially purified enzyme, whole cellstransformed with gene(s) encoding the enzyme, as cell extracts and/orlysates of such cells, and/or as an enzyme immobilized on a solidsupport. Whole cells transformed with gene(s) encoding the engineeredimine reductase enzyme or cell extracts, lysates thereof, and isolatedenzymes may be employed in a variety of different forms, including solid(e.g., lyophilized, spray-dried, and the like) or semisolid (e.g., acrude paste). The cell extracts or cell lysates may be partiallypurified by precipitation (ammonium sulfate, polyethyleneimine, heattreatment or the like, followed by a desalting procedure prior tolyophilization (e.g., ultrafiltration, dialysis, and the like). Any ofthe enzyme preparations (including whole cell preparations) may bestabilized by cross-linking using known cross-linking agents, such as,for example, glutaraldehyde or immobilization to a solid phase (e.g.,Eupergit C, and the like).

The gene(s) encoding the engineered imine reductase polypeptides can betransformed into host cell separately or together into the same hostcell. For example, in some embodiments one set of host cells can betransformed with gene(s) encoding one engineered imine reductasepolypeptide and another set can be transformed with gene(s) encodinganother engineered imine reductase polypeptide. Both sets of transformedcells can be utilized together in the reaction mixture in the form ofwhole cells, or in the form of lysates or extracts derived therefrom. Inother embodiments, a host cell can be transformed with gene(s) encodingmultiple engineered imine reductase polypeptide. In some embodiments theengineered polypeptides can be expressed in the form of secretedpolypeptides and the culture medium containing the secreted polypeptidescan be used for the imine reductase reaction.

The improved activity and/or stereoselectivity of the engineered iminereductase polypeptides disclosed herein provides for processes whereinhigher percentage conversion can be achieved with lower concentrationsof the engineered polypeptide. In some embodiments of the process, thesuitable reaction conditions comprise an engineered polypeptide amountof about 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 20% (w/w), 30% (w/w),40% (w/w), 50% (w/w), 75% (w/w), 100% (w/w) or more of substratecompound loading.

In some embodiments, the engineered polypeptides are present at about0.01 g/L to about 50 g/L; about 0.05 g/L to about 50 g/L; about 0.1 g/Lto about 40 g/L; about 1 g/L to about 40 g/L; about 2 g/L to about 40g/L; about 5 g/L to about 40 g/L; about 5 g/L to about 30 g/L; about 0.1g/L to about 10 g/L; about 0.5 g/L to about 10 g/L; about 1 g/L to about10 g/L; about 0.1 g/L to about 5 g/L; about 0.5 g/L to about 5 g/L; orabout 0.1 g/L to about 2 g/L. In some embodiments, the imine reductasepolypeptide is present at about 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.2 g/L,0.5 g/L, 1, 2 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35g/L, 40 g/L, or 50 g/L.

During the course of the reactions, the pH of the reaction mixture maychange. The pH of the reaction mixture may be maintained at a desired pHor within a desired pH range. This may be done by the addition of anacid or a base, before and/or during the course of the reaction.Alternatively, the pH may be controlled by using a buffer. Accordingly,in some embodiments, the reaction condition comprises a buffer. Suitablebuffers to maintain desired pH ranges are known in the art and include,by way of example and not limitation, borate, phosphate,2-(N-morpholino)ethanesulfonic acid (MES),3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine,and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), and the like. Insome embodiments, the buffer is phosphate. In some embodiments of theprocess, the suitable reaction conditions comprise a buffer (e.g.,phosphate) concentration is from about 0.01 to about 0.4 M, 0.05 toabout 0.4 M, 0.1 to about 0.3 M, or about 0.1 to about 0.2 M. In someembodiments, the reaction condition comprises a buffer (e.g., phosphate)concentration of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.07, 0.1, 0.12,0.14, 0.16, 0.18, 0.2, 0.3, or 0.4 M. In some embodiments, the reactionconditions comprise water as a suitable solvent with no buffer present.

In the embodiments of the process, the reaction conditions can comprisea suitable pH. The desired pH or desired pH range can be maintained byuse of an acid or base, an appropriate buffer, or a combination ofbuffering and acid or base addition. The pH of the reaction mixture canbe controlled before and/or during the course of the reaction. In someembodiments, the suitable reaction conditions comprise a solution pHfrom about 4 to about 10, pH from about 5 to about 10, pH from about 7to about 11, pH from about 8 to about 10, pH from about 6 to about 8. Insome embodiments, the reaction conditions comprise a solution pH ofabout 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

In the embodiments of the processes herein, a suitable temperature canbe used for the reaction conditions, for example, taking intoconsideration the increase in reaction rate at higher temperatures, andthe activity of the enzyme during the reaction time period. Accordingly,in some embodiments, the suitable reaction conditions comprise atemperature of from about 10° C. to about 80° C., about 10° C. to about70° C., about 15° C. to about 65° C., about 20° C. to about 60° C.,about 20° C. to about 55° C., about 25° C. to about 55° C., or about 30°C. to about 50° C. In some embodiments, the suitable reaction conditionscomprise a temperature of about 10° C., 15° C., 20° C., 25° C., 30° C.,35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C.,or 80° C. In some embodiments, the temperature during the enzymaticreaction can be maintained at a specific temperature throughout thecourse of the reaction. In some embodiments, the temperature during theenzymatic reaction can be adjusted over a temperature profile during thecourse of the reaction.

The processes of the invention are generally carried out in a solvent.Suitable solvents include water, aqueous buffer solutions, organicsolvents, polymeric solvents, and/or co-solvent systems, which generallycomprise aqueous solvents, organic solvents and/or polymeric solvents.The aqueous solvent (water or aqueous co-solvent system) may bepH-buffered or unbuffered. In some embodiments, the processes using theengineered imine reductase polypeptides can be carried out in an aqueousco-solvent system comprising an organic solvent (e.g., ethanol,isopropanol (IPA), dimethyl sulfoxide (DMSO), dimethylformamide (DMF),dimethylacetamide (DMAc), N-methyl-pyrrolidone (NMP), ethyl acetate,butyl acetate, 1-octanol, heptane, octane, methyl t butyl ether (MTBE),toluene, and the like), ionic or polar solvents (e.g.,1-ethyl-4-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium hexafluorophosphate, glycerol, polyethyleneglycol (PEG), and the like). In some embodiments, the co-solvent can bea polar solvent, such as a polyol, dimethylsulfoxide (DMSO), or loweralcohol. The non-aqueous co-solvent component of an aqueous co-solventsystem may be miscible with the aqueous component, providing a singleliquid phase, or may be partly miscible or immiscible with the aqueouscomponent, providing two liquid phases. Exemplary aqueous co-solventsystems can comprise water and one or more co-solvents selected from anorganic solvent, polar solvent, and polyol solvent. In general, theco-solvent component of an aqueous co-solvent system is chosen such thatit does not adversely inactivate the imine reductase enzyme under thereaction conditions. Appropriate co-solvent systems can be readilyidentified by measuring the enzymatic activity of the specifiedengineered imine reductase enzyme with a defined substrate of interestin the candidate solvent system, utilizing an enzyme activity assay,such as those described herein.

In some embodiments of the process, the suitable reaction conditionscomprise an aqueous co-solvent, where the co-solvent comprises DMSO atabout 1% to about 50% (v/v), about 1 to about 40% (v/v), about 2% toabout 40% (v/v), about 5% to about 30% (v/v), about 10% to about 30%(v/v), or about 10% to about 20% (v/v). In some embodiments of theprocess, the suitable reaction conditions can comprise an aqueousco-solvent comprising DMSO at about 1% (v/v), about 5% (v/v), about 10%(v/v), about 15% (v/v), about 20% (v/v), about 25% (v/v), about 30%(v/v), about 35% (v/v), about 40% (v/v), about 45% (v/v), or about 50%(v/v).

In some embodiments, the reaction conditions can comprise a surfactantfor stabilizing or enhancing the reaction. Surfactants can comprisenon-ionic, cationic, anionic and/or amphiphilic surfactants. Exemplarysurfactants, include by way of example and not limitations, nonylphenoxypolyethoxylethanol (NP40), Triton X-100,polyoxyethylene-stearylamine, cetyltrimethylammonium bromide, sodiumoleylamidosulfate, polyoxyethylene-sorbitanmonostearate,hexadecyldimethylamine, etc. Any surfactant that may stabilize orenhance the reaction may be employed. The concentration of thesurfactant to be employed in the reaction may be generally from 0.1 to50 mg/ml, particularly from 1 to 20 mg/ml.

In some embodiments, the reaction conditions can include an antifoamagent, which aid in reducing or preventing formation of foam in thereaction solution, such as when the reaction solutions are mixed orsparged. Anti-foam agents include non-polar oils (e.g., minerals,silicones, etc.), polar oils (e.g., fatty acids, alkyl amines, alkylamides, alkyl sulfates, etc.), and hydrophobic (e.g., treated silica,polypropylene, etc.), some of which also function as surfactants.Exemplary anti-foam agents include, Y-30® (Dow Corning), poly-glycolcopolymers, oxy/ethoxylated alcohols, and polydimethylsiloxanes. In someembodiments, the anti-foam can be present at about 0.001% (v/v) to about5% (v/v), about 0.01% (v/v) to about 5% (v/v), about 0.1% (v/v) to about5% (v/v), or about 0.1% (v/v) to about 2% (v/v). In some embodiments,the anti-foam agent can be present at about 0.001% (v/v), about 0.01%(v/v), about 0.1% (v/v), about 0.5% (v/v), about 1% (v/v), about 2%(v/v), about 3% (v/v), about 4% (v/v), or about 5% (v/v) or more asdesirable to promote the reaction.

The quantities of reactants used in the imine reductase reaction willgenerally vary depending on the quantities of product desired, andconcomitantly the amount of imine reductase substrate employed. Thosehaving ordinary skill in the art will readily understand how to varythese quantities to tailor them to the desired level of productivity andscale of production.

In some embodiments, the order of addition of reactants is not critical.The reactants may be added together at the same time to a solvent (e.g.,monophasic solvent, biphasic aqueous co-solvent system, and the like),or alternatively, some of the reactants may be added separately, andsome together at different time points. For example, the cofactor,co-substrate, imine reductase, and substrate may be added first to thesolvent.

The solid reactants (e.g., enzyme, salts, etc.) may be provided to thereaction in a variety of different forms, including powder (e.g.,lyophilized, spray dried, and the like), solution, emulsion, suspension,and the like. The reactants can be readily lyophilized or spray driedusing methods and equipment that are known to those having ordinaryskill in the art. For example, the protein solution can be frozen at−80° C. in small aliquots, then added to a pre-chilled lyophilizationchamber, followed by the application of a vacuum.

For improved mixing efficiency when an aqueous co-solvent system isused, the imine reductase, and cofactor may be added and mixed into theaqueous phase first. The organic phase may then be added and mixed in,followed by addition of the imine reductase substrate and co-substrate.Alternatively, the imine reductase substrate may be premixed in theorganic phase, prior to addition to the aqueous phase.

The imine reductase reaction is generally allowed to proceed untilfurther conversion of substrates to product does not changesignificantly with reaction time, e.g., less than 10% of substratesbeing converted, or less than 5% of substrates being converted). In someembodiments, the reaction is allowed to proceed until there is completeor near complete conversion of substrates to product. Transformation ofsubstrates to product can be monitored using known methods by detectingsubstrate and/or product, with or without derivatization. Suitableanalytical methods include gas chromatography, HPLC, and the like.

In some embodiments of the process, the suitable reaction conditionscomprise a loading of substrates of at least about 5 g/L, 10 g/L, 20g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L, or more, andwherein the method results in at least about 50%, 60%, 70%, 80%, 90%,95% or greater conversion of substrate compounds to product compound inabout 48 h or less, in about 36 h or less, or in about 24 h or less.

The engineered imine reductase polypeptides of the present inventionwhen used in the process under suitable reaction conditions result in adiastereomeric excess of the desired secondary or tertiary amine productin at least 90%, 95%, 96%, 97%, 98%, 99%, or greater. In someembodiments, no detectable amount of the undesired diastereomericsecondary or tertiary amine product is formed.

In further embodiments of the processes for converting substratecompounds to amine product compound using the engineered imine reductasepolypeptides, the suitable reaction conditions can comprise initialsubstrate loadings to the reaction solution which is then contacted bythe polypeptide. This reaction solution is the further supplemented withadditional substrate compounds as a continuous or batchwise additionover time at a rate of at least about 1 g/L/h, at least about 2 g/L/h,at least about 4 g/L/h, at least about 6 g/L/h, or higher. Thus,according to these suitable reaction conditions, polypeptide is added toa solution having initial ketone and amine substrate loadings of each atleast about 20 g/L, 30 g/L, or 40 g/L. This addition of polypeptide isthen followed by continuous addition of further ketone and aminesubstrates to the solution at a rate of about 2 g/L/h, 4 g/L/h, or 6g/L/h until a much higher final substrate loading of each at least about30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L, 150 g/L, 200 g/L ormore, is reached. Accordingly, in some embodiments of the process, thesuitable reaction conditions comprise addition of the polypeptide to asolution having initial substrate loadings of each at least about 20g/L, 30 g/L, or 40 g/L followed by addition of further ketone and aminesubstrates to the solution at a rate of about 2 g/L/h, 4 g/L/h, or 6g/L/h until a final substrate loading of at least about 30 g/L, 40 g/L,50 g/L, 60 g/L, 70 g/L, 100 g/L or more, is reached. These substratesupplementation reaction conditions allow for higher substrate loadingsto be achieved while maintaining high rates of conversion of substratesto amine product of at least about 50%, 60%, 70%, 80%, 90% or greaterconversion.

In some embodiments of the processes, the reaction using an engineeredimine reductase polypeptide can comprise the following suitable reactionconditions: (a) substrate loading at about 5 g/L to 30 g/L; (b) about0.1 g/L to 10 g/L of the engineered polypeptide; (c) about 19 g/L (0.13M) to 57 g/L (0.39 M) of α-ketoglutarate; (d) about 14 g/L (0.08 M) to63 g/L (0.36 M) ascorbic acid; (e) about 1.5 g/L (3.8 mM) to 4.5 g/L(11.5 mM) of FeSO₄; (f) a pH of about 6 to 9; (g) temperature of about20° to 50° C.; and (h) reaction time of 2-24 hrs.

In some embodiments of the processes, the reaction using an engineeredimine reductase polypeptide can comprise the following suitable reactionconditions: (a) substrate loading at about 10 g/L to 100 g/L; (b) about1 g/L to about 50 g/L of engineered polypeptide; (c) NADH or NADPHloading at about 0.1 g/L to about 5 g/L; (d) pH of about 6 to 10; (g)temperature of about 20° to 50° C.; and (h) reaction time of 6 to 120hrs.

In some embodiments, additional reaction components or additionaltechniques carried out to supplement the reaction conditions. These caninclude taking measures to stabilize or prevent inactivation of theenzyme, reduce product inhibition, shift reaction equilibrium to amineproduct formation.

In further embodiments, any of the above described process for theconversion of substrate compound to product compound can furthercomprise one or more steps selected from: extraction; isolation;purification; and crystallization of product compound. Methods,techniques, and protocols for extracting, isolating, purifying, and/orcrystallizing the amine product from biocatalytic reaction mixturesproduced by the above disclosed methods are known to the ordinaryartisan and/or accessed through routine experimentation. Additionally,illustrative methods are provided in the Examples below.

Various features and embodiments of the invention are illustrated in thefollowing representative examples, which are intended to beillustrative, and not limiting.

EXPERIMENTAL

The following examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present invention.

In the experimental disclosure below, the following abbreviations apply:ppm (parts per million); M (molar); mM (millimolar), uM and μM(micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg(milligrams); ug and ng (micrograms); L and 1 (liter); ml and mL(milliliter); cm (centimeters); mm (millimeters); um and μm(micrometers); sec (seconds); min(s) (minute(s)); h(s) and hr(s)(hour(s)); U (units); MW (molecular weight); rpm (rotations per minute);psi and PSI (pounds per square inch); ° C. (degrees Centigrade); RT andrt (room temperature); CAM and cam (chloramphenicol); DMSO(dimethylsulfoxide); PMBS (polymyxin B sulfate); IPTG (isopropylβ-D-1-thiogalactopyranoside); GDH (glucose dehydrogenase); FDH (formatedehydrogenase); LB (Luria broth); TB (terrific broth); SFP (shake flaskpowder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA(ribonucleic acid); E. coli W3110 (commonly used laboratory E. colistrain, available from the Coli Genetic Stock Center [CGSC], New Haven,Conn.); HTP (high throughput); HPLC (high performance liquidchromatography); FIOPC (fold improvements over positive control);Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.; Difco (Difco Laboratories,BD Diagnostic Systems, Detroit, Mich.); Agilent (Agilent Technologies,Inc., Santa Clara, Calif.); Corning (Corning, Inc., Palo Alto, Calif.);Dow Corning (Dow Corning, Corp., Midland, Mich.); and Gene Oracle (GeneOracle, Inc., Mountain View, Calif.).

Example 1 Production of Engineered Polypeptides Derived from CENDHHaving Imine Reductase Activity

The engineered imine reductase polypeptides of the even-numberedsequences of SEQ ID NOS:4-1300 were produced in E. coli W3110 under thecontrol of the lac promoter. Enzyme preparations for the HTP assays usedin the directed evolution of the engineered polypeptides were made asfollows.

High-Throughput (HTP) Growth, Expression, and Lysate Preparation.

Cells were picked and grown overnight in LB media containing 1% glucoseand 30 μg/mL CAM, 30° C., 200 rpm, 85% humidity. Then, 20 μL, ofovernight growth were transferred to a deep well plate containing 380μL, TB growth media containing 30 μg/mL CAM, and allowed to grow at 30°C., 250 rpm, 85% humidity. Expression of the imine reductase gene wasinduced by addition of IPTG to a final concentration of 1 mM when theOD₆₀₀ of the culture is 0.6 to 0.8. Incubation was then continuedovernight (˜18 h). Cell cultures were centrifuged at 4000 rpm, 4° C. for10 min, and the media discarded. The cell pellets were stored at −80° C.and used to prepare lysates for the HTP reactions as follows. Lysisbuffer containing 1 g/L lysozyme and 1 g/L PMBS was prepared in 0.1 Mphosphate buffer, pH 8.0. Cell pellets in 96 well plates were lysed in2504 lysis buffer, with medium-speed shaking for 2 hrs on a titre-plateshaker at room temperature. The plates then were centrifuged at 4000 rpmfor 20 mins at 4° C. and the clear supernatant was used as the clearlysate in the HTP assay reaction.

Production of Shake Flask Powders (SFP):

Shake-flask procedures can be used to generate engineered iminereductase polypeptide shake-flask powders (SFP) useful for secondaryscreening assays and/or find use in the biocatalytic processes disclosedherein. Shake flask powder (SFP) preparation of enzymes provides a morepurified preparation (e.g., up to 30% of total protein) of theengineered enzyme as compared to the cell lysate used in HTP assays and,among other things, allows for the use of more concentrated enzymesolutions. A single colony of E. coli containing a plasmid encoding anengineered polypeptide of interest is inoculated into 50 mL LuriaBertani broth containing 30 μg/ml chloramphenicol and 1% glucose. Cellsare grown overnight (at least 16 hours) in an incubator at 30° C. withshaking at 250 rpm. The culture is diluted into 250 mL Terrific Broth(12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65 mMpotassium phosphate, pH 7.0, 1 mM MgSO₄) containing 30 μg/ml CAM, in a 1L flask to an optical density of 600 nm (OD₆₀₀) of 0.2 and allowed togrow at 30° C. Expression of the imine reductase gene is induced byaddition of IPTG to a final concentration of 1 mM when the OD₆₀₀ of theculture is 0.6 to 0.8. Incubation is then continued overnight (at least16 hours). Cells are harvested by centrifugation (4000 rpm, 20 min, 4°C.) and the supernatant discarded. The cell pellet is washed byresuspension with an equal volume of cold (4° C.) 50 mM potassiumphosphate buffer, pH 8.0, and harvested by centrifugation as above. Thewashed cells are weighed and resuspended in 6 ml of cold 50 mM potassiumphosphate buffer, pH 8.0, per 1 g cell pellet. The resuspended cells arelysed using a 110L Microfluidizer (Microfluidics, Newton, Mass.). Celldebris is removed by centrifugation (10,000 rpm, 60 minutes, 4° C.). Theclear lysate supernatant is collected and stored at −20° C.Lyophilization of frozen clear lysate provides a dry shake-flask powderof crude engineered polypeptide. Alternatively, the cell pellet (beforeor after washing) can be stored at 4° C. or −80° C.

Example 2 Evolution and Screening of Engineered Polypeptides Derivedfrom SEQ ID NO:4 for Improved Stability and Imine Reductase Activity inPreparing Compounds 3a and 3b

The engineered polypeptide having imine reductase activity of SEQ IDNO:4 was used to generate the further engineered polypeptides of Tables3A-3C. These polypeptides have improved imine reductase activity (e.g.,% conversion of ketone substrate compound (1a) to product), as comparedto SEQ ID NO:4. The engineered polypeptide having imine reductaseactivity of SEQ ID NO:6 was then used to generate the further engineeredpolypeptides of Tables 3D-3F. These polypeptides have improved iminereductase activity (e.g., % conversion of ketone substrate compound (1b)to product), as compared to SEQ ID NO:6.

These engineered polypeptides, having the amino acid sequences ofeven-numbered sequence identifiers SEQ ID NOS:10-358, were generatedfrom the “backbone” amino acid sequence of SEQ ID NO:4 or 6 usingdirected evolution methods as described above together with the HTPassay and analytical methods noted in Table 4A and described furtherbelow.

Directed evolution began with the polynucleotides of SEQ ID NO:3 and 5,which encode the engineered polypeptides of SEQ ID NO:4 and 6,respectively, as the starting “backbone” gene sequences. Libraries ofengineered polypeptides were generated using various well-knowntechniques (e.g., saturation mutagenesis, recombination of previouslyidentified beneficial amino acid differences) and screened using HTPassay and analysis methods that measured the ability of the engineeredpolypeptides to carry out one or more of the catalytic reactions (A orB) shown in Table 2. After screening, the engineered polypeptide(s)showing the most improvement over the starting backbone sequence (or“control” sequence) were used as backbone sequences for the constructionof further libraries, and the screening process was repeated to furtherevolve the polypeptides for the desired activity.

The enzyme assay reaction was carried out in a total volume of 100 μL ina 96-well plate format. The assay reaction was initiated by adding thefollowing to each well containing 554 of the lysate: (i) 104 of GDHcofactor recycling pre-mix (pre-mix contains 10 g/L glucose, 30 g/LNAD+, 10 g/L GDH-105); (ii) 204 of amine substrate stock solution (500mM in 0.1M potassium phosphate buffer, pH adjusted to 8.0); and 154ketone substrate stock solution (333 mM in DMSO). The resulting assayreaction included 50 mM ketone substrate compound (1a or 1b), 100 mMamine substrate (2a or 2b), 55.5 mM glucose, 3 g/L NAD+, 1 g/L GDH-105,85 mM potassium phosphate, pH 8.0, 15% (v/v) DMSO. The reaction platewas heat-sealed and shaken at 400 rpm overnight (16-24 h) at 44° C. Eachreaction mixture was quenched by adding 1004 CH₃CN with 0.1% formicacid, shaken, and centrifuged at 4000 rpm, 4° C. for 10 mM. 104 of thequenched mixture was diluted 20 fold in CH₃CN/H20 (50/50) with 0.05%formic acid with mixing. 104 of this diluted mixture was then furtherdiluted in 1904 CH₃CN/H₂O (50/50) with 0.05% formic acid for a total 800fold diluted mixtures. These mixtures then were analyzed for productcompound (3a or 3b) formation by LC-MS in MRM mode as described in thetables below.

Instrument Agilent HPLC 1200 series, AB Sciex API 2000 Column OnyxMonolithic C8, 100 × 4.6 mm with Onyx monolithic C18 guard cartridge(Phenomenex) Mobile Phase Gradient (A: 0.1% formic acid in water; B:0.1% formic acid in MeCN) Time (min) % B 0.0.-0.8 25   2.0-2.75 902.85-5.0  25 Flow Rate 0.8 mL/min Run time 5 min Peak Retention TimesCompound (3a): 3.5 min Column Temperature Room Temperature InjectionVolume 10 μL MS Detection API 2000; MRM 347/232 (Product 3a) MSConditions MODE: MRM; CUR: 30; IS: 4500; CAD 12: TEM: 550° C.; GS1: 60;GS2: 60; DP: 31; FP: 350; EP: 10; CE: 25; CXP: 4; DT: 100 ms

LC-MS Analysis for Amine Product Compound 3a (Table 4A):

The HTP assay mixtures prepared as noted in Example 2 above wereanalyzed for formation of the product compound (3a) by LC-MS in MRM modeusing the MRM transition: 347/232. Additional relevant LC-MSinstrumental parameters and conditions were as shown below.

LC-MS Analysis for Amine Product Compound 3b (Table 4b):

The HTP assay mixtures prepared as noted in Example 2 above wereanalyzed for formation of the product compound (3b) by LC-MS in MRM modeusing the MRM transition: 242/168. Additional relevant LC-MSinstrumental parameters and conditions were as shown below.

Instrument Agilent HPLC 1200 series, AB Sciex API 2000 Column OnyxMonolithic C8, 100 × 4.6 mm with Onyx monolithic C18 guard cartridge(Phenomenex) Mobile Phase Gradient (A: 0.1% formic acid in water; B:0.1% formic acid in MeCN) Time (min) % B 0.0.-0.8 25   2.0-2.75 902.85-4.0  25 Flow Rate 0.8 mL/min Run time 4 min Peak Retention TimesCompound (3b): 2.5 min Column Temperature Room Temperature InjectionVolume 10 μL MS Detection API 2000; MRM 242/168 (Product 3b) MSConditions MODE: MRM; CUR: 30; IS: 4500; CAD 12: TEM: 550° C.; GS1: 60;GS2: 60; DP: 31; FP: 350; EP: 10; CE: 25; CXP: 4; DT: 100 ms

Example 3 Evolution and Screening of Engineered Polypeptides Derivedfrom SEQ ID NO:8 for Improved Stability and Imine Reductase Activity inPreparing Compound 3c, 3d, 3e, 3f and 3g

The engineered polypeptide having imine reductase activity of SEQ IDNO:8 was used to generate the further engineered polypeptides of Tables3G-3M. These polypeptides have further improved imine reductase activity(e.g., % conversion of ketone substrate compound (1c or 1d) to product),as indicated in these Tables. These engineered polypeptides, which havethe amino acid sequences of even-numbered sequence identifiers SEQ IDNOS:358-1300, were generated from the “backbone” amino acid sequence ofSEQ ID NO:8, using directed evolution methods as described above, aswell as the HTP assay and analytical methods indicated in Tables 4C, 4Dand 4E, and described further below.

Directed evolution began with the polynucleotide of SEQ ID NO:7, whichencodes the engineered polypeptide of SEQ ID NO:8, as the starting“backbone” gene sequence. Libraries of engineered polypeptides weregenerated using various well-known techniques (e.g., saturationmutagenesis, recombination of previously identified beneficial aminoacid differences) and screened using HTP assay and analysis methods thatmeasured the ability of the engineered polypeptides to carry out one ormore of the catalytic reactions (C, D, E, F and/or G) shown in Table 2.After screening, the engineered polypeptide(s) showing the mostimprovement over the starting backbone sequence (or “control” sequence)were used as backbone sequences for the construction of furtherlibraries, and the screening process repeated to evolve the polypeptidefor the desired activity.

The enzyme assay reaction was carried out in a total volume of 100 μL,in a 96-well plate format. The assay reaction was initiated by addingthe following to each well containing 55 μL, of the lysate: (i) 10 μL,of GDH cofactor recycling pre-mix (pre-mix contains 100 g/L glucose, 30g/L NAD+, 10 g/L GDH-105); (ii) 20 μL, of amine substrate stock solution(250 mM in 0.1M potassium phosphate buffer, pH adjusted to 8.0); and 15μL, ketone substrate stock solution (333 mM compound in DMSO). Theresulting assay reaction included 50 mM ketone substrate compound (1c,or 1d), 100 mM or 50 mM amine substrate (2c or 2d), 55.5 mM glucose, 3g/L NAD+, 1 g/L GDH-105, 85 mM potassium phosphate, pH 8.0, 15% (v/v)DMSO. The reaction plate was heat-sealed and shaken at 400 rpm overnight(16-24 h) at 44° C. Each reaction mixture was quenched by adding 100 μL,CH₃CN with 0.1% formic acid, shaken, and centrifuged at 4000 rpm and 4°C. for 10 min. 10 μL, of the quenched mixture was diluted 10 fold inCH₃CN/H₂O (50/50) with 0.05% formic acid with mixing, for a total 100fold diluted mixtures. These mixtures then were analyzed for productcompound (3c, 3d, 3e, 3f and/or 3g) formation by LC-MS in MRM mode asdescribed in the tables below.

LC-MS Analysis for Amine Product Compounds (3c) (Table 4c):

The HTP assay mixtures prepared as noted in Example 3 above wereanalyzed for formation of the product compound (3c) by LC-MS in MRM modeusing the MRM transition: 300.3/190.2. Additional relevant LC-MSinstrumental parameters and conditions were used as indicated below.

Instrument Agilent HPLC 1200 series, AB Sciex API 2000 Column OnyxMonolithic C8 100 × 4.6 mm (Phenomenex) with Onyx Monolithic C18 guardcartridge. Mobile Phase Gradient (A: 0.1% formic acid in water; B: 0.1%formic acid in MeCN) Time (min) % B 0.0.-0.8 25   2.0-2.75 90 2.85-3.8 25 Flow Rate 0.8 mL/min Run time 3.8 min Peak Retention Times Compound(3c): 2.38 min Column Temperature Room Temperature Injection Volume 10μL MS Detection API 2000; MRM MS Conditions MODE: MRM 300.3/190.2; CUR:30; IS: 4500; CAD 12: TEM: 550° C.; GS1: 60; GS2: 60; DP: 31; FP: 350;EP: 10; CE: 30; CXP: 4; DT: 200 ms

LC-MS Analysis for Amine Product Compounds 3d (Table 4D):

The HTP assay mixtures prepared as noted in Example 3 above wereanalyzed for formation of the product compound (3d) by LC-MS in MRM modeusing the MRM transition: 385.3/329.4. Additional relevant LC-MSinstrumental parameters and conditions were used as indicated below.

Instrument Agilent HPLC 1200 series, AB Sciex API 2000 Column OnyxMonolithic C8 100 × 4.6 mm (Phenomenex) with Onyx Monolithic C18 guardcartridge. Mobile Phase Gradient (A: 0.1% formic acid in water; B: 0.1%formic acid in MeCN) Time (min) % B 0.0.-0.8 25   2.0-2.75 90 2.85-5.1 25 Flow Rate 0.8 mL/min Run time 5.1 min Peak Retention Times Compound(3d): 4.39 Column Temperature Room Temperature Injection Volume 10 μL MSDetection API 2000; MRM MS Conditions MODE: MRM 385.3/329.4; CUR: 30;IS: 4500; CAD 12: TEM: 550° C.; GS1: 60; GS2: 60; DP: 31; FP: 350; EP:10; CE: 40; CXP: 4; DT: 200 ms

LC-MS Analysis for Amine Product Compounds (3e, 3f and 3g) (Table 4E):

The HTP assay mixtures prepared as noted above in Example 3 wereanalyzed for formation of the product compounds 3f, 3g and 3e (sum of 3fand 3g), by LC-MS in MS2 mode using 499.2 mass unit detection.Additional relevant LC-MS instrumental parameters and conditions wereused as indicated below.

Instrument Agilent HPLC 1200 series, AB Sciex API 2000 Column OnyxMonolithic C8 100 × 4.6 mm (Phenomenex) with Onyx Monolithic C18 guardcartridge. Mobile Phase Gradient (A: 0.1% formic acid in water; B: 0.1%formic acid in MeCN) Time (min) % B 0.0.-0.8 35   2.0-2.75 90 2.85-6.1 35 Flow Rate 0.8 mL/min Run time 6.1 min Peak Retention Times Compound(3f): 3.59 min Compound (3g): 4.19 min Column Temperature RoomTemperature Injection Volume 10 μL MS Detection API 2000; For bothstereoisomers - MS2: Product of 499.2 Da, scan fragments 102-502 Da MSConditions MODE: MS2; CUR: 30; IS: 4500; CAD 12: TEM: 550° C.; GS1: 60;GS2: 60; DP: 31; FP: 350; EP: 10; CE: 40; CXP: 4; DT: 200 ms

What is claimed is:
 1. An engineered polynucleotide encoding anengineered polypeptide comprising an amino acid sequence with at least90% sequence identity to the reference sequence of SEQ ID NO:8, whereinthe alanine at the position corresponding to amino acid 239 of SEQ IDNO:8 is substituted with another amino acid in the amino acid sequenceof the engineered polypeptide, and wherein the polypeptide has iminereductase activity.
 2. An engineered polynucleotide encoding anengineered polypeptide comprising an amino acid sequence with at least90% sequence identity to the reference sequence of SEQ ID NO:8, whereinthe isoleucine at the position corresponding to amino acid 330 of SEQ IDNO:8 is substituted with another amino acid in the amino acid sequenceof the engineered polypeptide, wherein the amino acid sequence of theengineered polypeptide has a valine, threonine, tryptophan, glutamicacid, or tyrosine at the position corresponding to amino acid 283 of SEQID NO:8, and wherein the engineered polypeptide has imine reductaseactivity.
 3. The engineered polynucleotide encoding the engineeredpolypeptide of claim 2, wherein the amino acid sequence of theengineered polypeptide comprises at least one residue difference ascompared to the reference sequence of SEQ ID NO:4, wherein the at leastone residue difference is at a residue selected from the groupconsisting of 198, 153, 167, 265, 262, 108, 234, 284, 282, 220, 272,256, 267, 242, 281, 197, 277, 224, and 143, and wherein the residuenumber corresponds to the amino acid sequence of SEQ ID NO:4.
 4. Theengineered polynucleotide encoding the engineered polypeptide of claim2, wherein the amino acid sequence of the engineered polypeptidecomprises at least one residue difference as compared to the referencesequence of SEQ ID NO:6, wherein the at least one residue difference isat a residue selected from the group consisting of 262, 9, 259, 220,267, 153, 279, 200, 224, 256, 137, 143, 260, 261, 154, 276, and 185, andwherein the residue number corresponds to the amino acid sequence of SEQID NO:6.
 5. The engineered polynucleotide encoding the engineeredpolypeptide of claim 2, wherein the amino acid sequence of theengineered polypeptide further comprises at least one residue differenceas compared to the reference sequence of SEQ ID NO:8, wherein the atleast one residue difference is at a residue selected from the groupconsisting of 12, 31, 37, 44, 46, 48, 55, 56, 65, 82, 93, 98, 108, 137,138, 141, 142, 143, 153, 154, 155, 156, 159, 166, 168, 173, 177, 178,184, 185, 195, 197, 199, 200, 201, 203, 205, 206, 210, 213, 215, 216,218, 220, 221, 223, 226, 234, 239, 242, 245, 251, 253, 256, 257, 259,260, 261, 262, 263, 265, 266, 267, 268, 269, 273, 274, 277, 278, 279,280, 281, 282, 284, 285, 289, 290, 291, 292, 293, 294, and 349, andwherein the residue number corresponds to the amino acid sequence of SEQID NO:8.
 6. The engineered polynucleotide encoding the engineeredpolypeptide of claim 2, wherein the imine reductase activity comprisesconverting ketone substrate

and the amine substrate

to produce the amine product compound

under suitable reaction conditions.
 7. The engineered polynucleotideencoding the engineered polypeptide of claim 6, wherein the iminereductase activity in converting the ketone and the amine substratecompound to the amine product compound under suitable reactionconditions is increased at least 2-fold as compared to the correspondingactivity of an imine reductase polypeptide comprising the amino acidsequence of SEQ ID NO:4, 6, 12, 30, 126, 173, 360, or
 550. 8. Theengineered polynucleotide encoding the engineered polypeptide of claim1, wherein the amino acid sequence of the engineered polypeptidecomprises SEQ ID NO:1188.
 9. The engineered polynucleotide of claim 1,in which the polynucleotide comprises a nucleic acid sequence selectedfrom the group consisting of the odd-numbered sequence identifiers ofSEQ ID NOS:5-1299.
 10. The engineered polynucleotide of claim 2, inwhich the polynucleotide comprises a nucleic acid sequence selected fromthe group consisting of the odd-numbered sequence identifiers of SEQ IDNOS:5-1299.
 11. An expression vector comprising the engineeredpolynucleotide of claim
 1. 12. An expression vector comprising theengineered polynucleotide of claim
 2. 13. A host cell comprising theengineered polynucleotide of claim
 1. 14. A host cell comprising theengineered polynucleotide of claim
 2. 15. A host cell comprising theexpression vector of claim
 11. 16. A host cell comprising the expressionvector of claim
 12. 17. A method of preparing the engineered polypeptidehaving imine reductase activity, comprising culturing the host cell ofclaim 13, under conditions suitable for expression of the polypeptide,optionally further comprising isolating the engineered polypeptide.